WO2020213604A1 - 新規βアミラーゼ及びその利用・製造法 - Google Patents

新規βアミラーゼ及びその利用・製造法 Download PDF

Info

Publication number
WO2020213604A1
WO2020213604A1 PCT/JP2020/016434 JP2020016434W WO2020213604A1 WO 2020213604 A1 WO2020213604 A1 WO 2020213604A1 JP 2020016434 W JP2020016434 W JP 2020016434W WO 2020213604 A1 WO2020213604 A1 WO 2020213604A1
Authority
WO
WIPO (PCT)
Prior art keywords
amylase
seq
amino acid
dna
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2020/016434
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
順一 炭谷
修治 谷
川口　剛司
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University Public Corporation Osaka
Original Assignee
University Public Corporation Osaka
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Public Corporation Osaka filed Critical University Public Corporation Osaka
Priority to JP2021514172A priority Critical patent/JP7649547B2/ja
Priority to US17/603,836 priority patent/US12473542B2/en
Priority to CN202080028731.0A priority patent/CN114072519B/zh
Publication of WO2020213604A1 publication Critical patent/WO2020213604A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • 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; 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12P19/12—Disaccharides
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2523/00—Culture process characterised by temperature
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a novel ⁇ -amylase and a method for using and producing the novel ⁇ -amylase.
  • Starch is an important nutrient source for living organisms, and like cellulose, it is one of the many polysaccharides on the earth.
  • the structure of starch is composed of amylose in which glucose is linearly polymerized with ⁇ -1,4 bonds and amylopectin in which amylose is branched by ⁇ -1,6 bonds.
  • amylose When natural starch is suspended in water and heated, each molecule of amylose and amylopectin is dispersed to form a starch solution or starch paste.
  • starch having a natural crystal / grain structure is called raw starch. The crystal structure of raw starch is destroyed by heating and hydration.
  • Amylase is a general term for enzymes that hydrolyze the ⁇ -1,4 and ⁇ -1,6 bonds of amylose and amylopectin in this starch.
  • Amylase can be roughly classified into two types, endo-type amylase and exo-type amylase, depending on the mode of action.
  • ⁇ -Amylase is known as endoamylase and is an enzyme that randomly hydrolyzes the glucose chain of starch.
  • ⁇ -amylase is known as exo-type amylase, and is an enzyme in which glucose such as starch and glycogen acts on polysaccharides polymerized by ⁇ -1,4 bonds to decompose maltose (maltose) units from non-reducing ends.
  • ⁇ -amylase is used in maltose production and food processing due to its ability to produce maltose from starch.
  • Beta-amylase was found in sweet potatoes, wheat, barley, soybeans, etc., and has become known as an enzyme widely distributed in higher plants.
  • microbial-derived ⁇ -amylase does not exist for a long time, but ⁇ -amylase possessed by microorganisms has been discovered one after another since the 1970s (Non-Patent Document 1).
  • Microbial-derived ⁇ -amylase has been discovered one after another, but few have been put into practical use. This is because ⁇ -amylase derived from microorganisms reported so far has low heat resistance and production amount, and many of the producing bacteria are classified as pathogens and are not suitable for food use. Amano Enzyme Co., Ltd. barely sells ⁇ -amylase derived from Bacillus flexus as an industrial enzyme (Patent Document 1, Non-Patent Document 2). Although this ⁇ -amylase derived from Bacillus flexus has relatively high heat resistance compared to ⁇ -amylase derived from other microorganisms, it has lower heat resistance than soybean-derived enzyme, which has the highest heat resistance derived from plants, and has sufficient heat resistance.
  • soybean-derived enzymes having excellent heat resistance also have low decomposition activity on raw starch.
  • starch is industrially enzymatically decomposed with amylase, the starch is heated once, but from the viewpoint of energy saving in recent years, the raw starch is directly enzymatically reacted with high efficiency without heat gelatinization. It is hoped that it can be done.
  • the present invention thus provides the bacterium identified by accession number NITE BP-02937.
  • the present invention also provides ⁇ -amylase produced by the bacterium identified by accession number NITE BP-02937.
  • the present invention (i) The amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 12 or (ii) In the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 12, the amino acid sequence in which one to several amino acid residues are deleted, substituted, inserted, and / or added. (iii) Provided is ⁇ -amylase consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 12.
  • the threonine at the position corresponding to the amino acid number 4 is replaced with proline, or the threonine at the position corresponding to the amino acid number 15 is lysine or Provided is a ⁇ -amylase containing one or more substitutions selected from substitutions that change to arginine, or substitutions in which glutamine at the position corresponding to amino acid number 306 changes to aspartic acid.
  • the present invention (i) The amino acid sequence according to any one of SEQ ID NOs: 18 to 25 or (ii) In the amino acid sequence set forth in any one of SEQ ID NOs: 18 to 25, one to several amino acid residues are deleted, substituted, inserted, and / or added. (iii) Provided is ⁇ -amylase consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 18 to 25.
  • the present invention provides DNA encoding the above-mentioned ⁇ -amylase. Furthermore, the present invention is a DNA encoding a protein having ⁇ -amylase activity, wherein the DNA is (i) Nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 13 (ii) Nucleotide sequence 90% or more identical to the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 13 (iii) (i) or (ii) Provided is a DNA that hybridizes with the DNA represented by the base sequence represented by (1) under stringent conditions.
  • the present invention is a DNA encoding a protein having ⁇ -amylase activity, wherein the DNA is (i) The nucleotide sequence according to any one of SEQ ID NO: 26 to SEQ ID NO: 33 or (ii) DNA having any one of the base sequences having 90% or more sequence identity with the base sequence set forth in any one of SEQ ID NOs: 26 to 33, or (iii) Provided is a DNA that hybridizes with the DNA represented by the base sequence represented by (i) or (ii) under stringent conditions. Furthermore, the present invention provides DNA consisting of degenerate sequences of the nucleotide sequences represented by SEQ ID NO: 1, SEQ ID NO: 13 and SEQ ID NOs: 26 to 33.
  • the present invention provides a vector having the above DNA. Furthermore, the present invention provides a microorganism having a vector having the above DNA. Furthermore, the present invention provides a method for producing ⁇ -amylase, which comprises a step of extracting ⁇ -amylase from a culture of the above-mentioned bacteria or the above-mentioned microorganisms. Furthermore, the present invention provides a method for producing maltose, which comprises treating starch with the above-mentioned ⁇ -amylase. Furthermore, the present invention provides a method for producing maltose, which comprises treating starch with the above-mentioned bacteria or the above-mentioned microorganisms.
  • the present invention provides a method for producing maltose, which comprises treating raw starch with the above-mentioned ⁇ -amylase. Furthermore, the present invention provides a method for modifying foods using the above-mentioned bacteria, the above-mentioned ⁇ -amylase, or the above-mentioned microorganisms. Furthermore, the present invention provides an enzymatic agent using the above-mentioned bacteria, the above-mentioned ⁇ -amylase, or the above-mentioned microorganisms.
  • a novel ⁇ -amylase having no pathogenicity is provided.
  • the enzyme according to the present invention is ⁇ -amylase, which is an enzyme that decomposes ⁇ -1,4 glucoside bonds in glucose polysaccharides in starch in maltose units from non-reducing ends.
  • the ⁇ -amylase produced by the No. 58 strain has the following properties. (1)
  • the substrate for ⁇ -amylase of the present invention is not limited to starch.
  • the ⁇ -amylase of the present invention can react with, for example, amylose, amylopectin, glycogen, and dextrin.
  • the molecular weight of this enzyme is about 60,000 Da.
  • the optimum temperature of this enzyme is about 50 ° C. This enzyme exhibits 70% or more of the activity at 50 ° C.
  • the optimum pH of this enzyme is about 7.0. This enzyme shows a residual activity of 80% or more compared with the activity of pH 7.0 under the condition of reacting with the substrate at pH 6.0 to 9.0 and 37 ° C. for 10 minutes.
  • ⁇ -amylase of the present invention one to several amino acid residues are deleted or substituted in the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 12 or the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 12.
  • the inserted and / or added amino acid sequence, or ⁇ -amylase consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 12.
  • the amino acid sequence identity is preferably 95%, more preferably 99% or more.
  • the ⁇ -amylase of the present invention may be in the form of a fusion protein added to another protein as long as it has ⁇ -amylase activity, and is immobilized on a resin such as beads. It may be in shape.
  • the fusion protein include proteins bound to debranching enzyme, isoamylase, plulanase, glucosidase, glucoamylase, ⁇ -amylase, protease, lipase, phosphatase, xylanase, etc., histidine (His) tag for purification, and maltose-binding protein.
  • the form of a protein in which the (MBP) tag and glutathione S-transferase (GST) are fused is considered.
  • the substitution is preferably a conservative substitution.
  • Conservative substitution refers to a substitution in which the properties such as acidity and basicity of the replaced amino acid do not change. Specifically, substitution between Phe, Trp, Tyr, substitution between Leu, Ile, Val, substitution between Lys, Arg, His, substitution between Asp, Glu, substitution between Ser, Thr. Point to.
  • deletions at the N-terminal and / or C-terminal of the amino acid sequence, if the enzyme activity is equivalent to that of ⁇ -amylase having the amino acid sequence represented by SEQ ID NO: 2, the N-terminal and / or C of the amino acid sequence Not limited to the number of amino acids deleted at the terminus, such deletions can be easily obtained without trial and error.
  • the term "1 to several" as used herein means, for example, 1 to 9, preferably 1 to 8, more preferably 1 to 6, more preferably 1 to 5, more preferably 1 to 4, and more. The number may be preferably between 1 and 3, or more preferably between 1 and 2.
  • the substitution site is a substitution in which the threonine at amino acid number 4 of the amino acid sequence shown in SEQ ID NO: 2 is changed to proline (SEQ ID NO: 18), or a substitution in which the threonine at amino acid number 15 is changed to lysine (SEQ ID NO: 19).
  • Amino acid number 33 is changed to proline (SEQ ID NO: 22), or amino acid number 44 is changed to lysine (SEQ ID NO: 23), or amino acid number 44 is changed to arginine (sequence). If one or more of the substitutions (SEQ ID NO: 25) in which glutamine at amino acid number 335 is changed to aspartic acid is selected, ⁇ -amylase having further excellent heat resistance can be obtained. Further, the ⁇ -amylase of the present invention is one to several in the amino acid sequence set forth in any one of SEQ ID NOs: 18 to 25 or the amino acid sequence set forth in any one of SEQ ID NOs: 18 to 25.
  • the amino acid sequence identity is preferably 95%, more preferably 99% or more.
  • the same in the present invention is calculated as the ratio (percentage) of matching bases or amino acids at the corresponding positions between the two sequences when the two sequences to be compared are aligned so as to be the maximum match.
  • Analysis of amino acid sequences or comparison of the identity of two or more amino acid sequences can be performed using an analysis program commonly used by those skilled in the art. For example, GENETYX-WIN Ver.
  • NCBI http://www.ncbi.nlm.nih.gov/
  • BLAST search can be used for amino acid sequence identity search. .. Similar to the amino acid sequence, the base sequence can be analyzed and searched by an analysis program generally used by those skilled in the art.
  • the enzyme activity measurement according to the amylase of the present invention is not particularly limited as long as it is a method in which the enzyme decomposes the substrate and the saccharides produced by the decomposition can be quantitatively measured.
  • it can be measured by using a reducing sugar quantification method such as the Somogie-Nelson method.
  • the enzyme reaction product of amylase of the present invention may be detected by using a qualitative detection method when the purpose is only to detect the contained sugar, in addition to the quantitative detection method as described above. This is not particularly limited as long as the saccharides contained in the analysis target can be identified, but for example, a detection method using thin layer chromatography (TLC) can be considered.
  • TLC thin layer chromatography
  • the ⁇ -amylase of the present invention may have at least 80% activity as compared with the unheat-treated ⁇ -amylase even if the ⁇ -amylase is heat-treated at 60 ° C. for 10 minutes. Further, the enzyme may be heat-treated at 60 ° C. for 20, 40, 80 minutes and may have at least 80% activity as compared with unheat-treated ⁇ -amylase.
  • the ⁇ -amylase of the present invention may have a raw starch degrading activity.
  • the raw starch in the present invention is starch that has not been heat-treated. Unlike heat-gelatinized starch, raw starch has a crystal / grain structure. The crystal structure of raw starch is destroyed by heating and hydration.
  • the heating temperature varies depending on the type of raw starch, but for example, starch derived from wheat or corn has a high gelatinization temperature, and the gelatinization temperature needs to be heated to 80 ° C. or higher.
  • Starch which is the substrate of the enzyme, can be used for example, soybeans, red beans, barley, wheat, corn, rice, potatoes, sweet potatoes, kudzu, warabi, mung beans, soramame, lotus root, cassava, sago palm, katakuri, etc. Can be used.
  • Bacillus halosacchalovorans No. 58 (sometimes referred to simply as "No. 58 strain” in the present specification), which is a bacterium entrusted with the accession number NITE BP-02937.
  • the No. 58 strain is an aerobic bacterium, which has a rod-like cell morphology and is characterized by forming cream-colored colonies and forming spores. Since this strain is a non-pathogenic strain, it can be suitably used for foods and the like.
  • the ⁇ -amylase having the amino acid shown in SEQ ID NO: 2 according to the present invention is an enzyme obtained from the No. 58 strain.
  • the No. 58 strain has the DNA sequence represented by SEQ ID NO: 1 and ⁇ -amylase, which is a protein having the amino acid sequence of SEQ ID NO: 2.
  • the ⁇ -amylase of the present invention cultivates a bacterium having a DNA represented by SEQ ID NO: 1, such as strain No. 58, or a recombinant having a vector containing the sequence or incorporating the sequence.
  • a bacterium having a DNA represented by SEQ ID NO: 1 such as strain No. 58
  • a recombinant having a vector containing the sequence or incorporating the sequence can be obtained by Culturing of No. 58 strain or recombinant can be carried out by a method generally performed by those skilled in the art in amylase production.
  • the host microorganism can also be one commonly used by those skilled in the art.
  • the host microorganism is not particularly limited, and for example, non-pathogenic Escherichia coli, yeast, actinomycetes, algae, lactic acid bacteria, Bacillus subtilis and the like can be used.
  • the culture conditions can be appropriately adjusted according to the host and expression system.
  • the medium is not particularly limited as long as the culture target can grow.
  • the medium may be solid or liquid, but is preferably liquid.
  • the ⁇ -amylase gene is inserted into a vector for E. coli expression to transform E. coli.
  • the obtained transformants were inoculated into 2 ml of 2 ⁇ TY medium containing antibiotics as needed, and cultured with shaking at 30 ° C. for 24 hours to obtain bacterial cells.
  • ⁇ -amylase can be obtained in the crushed solution by sonication.
  • the solid medium for example, fruit residues such as bananas, apples and oranges, beans such as soybeans, and biomass derived from plant resources such as pomace of vegetable oil can be considered.
  • the culture is a mixture containing a substrate, a reaction product thereof, and cultured bacterial cells, which are cultured by adding a microorganism to a culture substrate.
  • Cultures include, but are not limited to, cultured microorganisms (inoculated microorganisms and microorganisms grown from the microorganisms), media, medium supplements / additives, and substances secreted from the cultured microorganisms.
  • the culture may be liquid or solid in whole or in part. If the culture is liquid, it is also called a culture solution.
  • the culture preferably comprises at least one of the culture microorganisms and secretions from the culture microorganisms.
  • the culture is preferably a culture medium.
  • the DNA of the present invention is a DNA encoding the above-mentioned ⁇ -amylase. Further, the DNA of the present invention is a DNA encoding a protein having ⁇ -amylase activity, and the DNA is (i) Nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 13. (ii) DNA having either 90% or more of the same base sequence as the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 13, or It may be a DNA that hybridizes with the DNA represented by the base sequence represented by (i) or (ii) under stringent conditions. The term "identical" with respect to the base sequence is as described above.
  • Stringent conditions are generally known by those skilled in the art, for example, in a commercially available hybridization solution ExpressHyb Hybridization Solution (Takara Bio), using conditions that hybridize at 68 ° C or using a DNA-fixed filter. After hybridization at about 65 ° C. in the presence of 0.7M to 1.0M NaCl, 0.1 to 2 times the concentration of SSC solution (1 ⁇ SSC solution: 150 mM NaCl, 15 mM sodium citrate, pH 7.0). Conditions such as washing at about 65 ° C. using The base sequence preferably has 95% or more, more preferably 99% or more, the same base sequence as the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 13.
  • substitution sites of the DNA sequence are, for example, a substitution in which the 97th adenine base of SEQ ID NO: 1 is changed to cytosine (SEQ ID NO: 26), a substitution in which the 131st cytosine base of SEQ ID NO: 1 is changed to an adenine base (SEQ ID NO: 27), and a sequence.
  • SEQ ID NO: 1 is replaced with a cytosine base
  • the 131st cytosine base is replaced with a guanine base
  • the 132nd adenine base is replaced with a cytosine base (SEQ ID NO: 28)
  • the 1003th cytosine base of SEQ ID NO: 1 is Substitution of guanine base with 1005th adenine base changed to cytosine base (SEQ ID NO: 29), or substitution of 10th adenine base of SEQ ID NO: 13 with cytosine (SEQ ID NO: 30), 44th cytosine of SEQ ID NO: 13
  • substitution in which the base is changed to an adenine base (SEQ ID NO: 31)
  • the 43rd adenine base in SEQ ID NO: 13 is changed to a cytosine base
  • the 44th sitosine base is changed to a guanine base
  • the 45th adenine base is changed to a cytosine base (
  • the DNA of the present invention is a DNA encoding a protein having ⁇ -amylase activity
  • the DNA is (i) The base sequence according to any one of SEQ ID NO: 26 to SEQ ID NO: 33, (ii) It has any of the base sequences having 90% or more, preferably 95% or more, more preferably 99% or more sequence identity with the base sequence shown in any one of SEQ ID NOs: 26 to 33.
  • DNA, or (iii) It may be a DNA that hybridizes with a DNA consisting of the base sequence represented by (i) or (ii) under stringent conditions.
  • the DNA of the present invention may be a DNA consisting of a degenerate sequence of the nucleotide sequences set forth in SEQ ID NO: 1, SEQ ID NO: 13 and SEQ ID NOs: 26 to 33.
  • degeneration means that there are a plurality of types of codons encoding one amino acid. Therefore, in the degenerate sequence, for example, when one amino acid in the degenerate sequence is arginine, the base sequence has six codons encoding arginine (CGU, CGC, CGA, CGG, AGA, AGG). It may be an array corresponding to any of the above.
  • the DNA of the present invention can be handled by using a method of genetic engineering generally performed by those skilled in the art.
  • the DNA sequence represented by SEQ ID NO: 1 may be obtained by PCR treatment using the corresponding primer, or may be obtained by total synthesis based on the sequence information.
  • the DNA of the present invention is also provided in the form of a vector having the DNA of the present invention.
  • the vector is generally used by those skilled in the art and is not limited as long as it can satisfy the purpose of incorporating this gene, but for example, pBluescript II SK (+) vector and pBluescript II SK (-) vector can be used.
  • the present invention provides a microorganism having a vector having the DNA of the present invention.
  • the host microorganism is not particularly limited as long as it is generally used as a vector carrier by those skilled in the art, and for example, non-pathogenic Escherichia coli, yeast, actinomycetes, algae, lactic acid bacteria, Bacillus subtilis and the like can be used. ..
  • the present invention also provides a method for producing ⁇ -amylase.
  • the ⁇ -amylase of the present invention may be obtained by isolating from a culture solution of a microorganism containing ⁇ -amylase.
  • the isolation method can be carried out by a method generally used by those skilled in the art.
  • the production method includes a step of culturing the cells and a step of extracting ⁇ -amylase from the culture or the cultured cells.
  • the step of culturing the cells is to cultivate the No. 58 strain or a recombinant microorganism containing the DNA of the present invention.
  • the host to be a recombinant is not particularly limited as long as it can be cultured and expresses a protein, and for example, non-pathogenic Escherichia coli, yeast, actinomycetes, algae, lactic acid bacteria, Bacillus subtilis and the like can be used.
  • the culture conditions can be appropriately adjusted according to the host and expression system.
  • the medium is not particularly limited as long as the culture target can grow.
  • the medium may be solid or liquid, but is preferably liquid.
  • the culturing method is not particularly limited as long as the culturing target can grow. For example, static culture, shaking culture, anaerobic culture and the like can be used. For example, when using No. 58 strain, the following conditions can be mentioned.
  • Examples of the medium used include liquid medium for amylase-producing bacteria, 2 ⁇ TY medium (listed in Tables 1 and 2 of Examples), LB medium, and Nutrient Agar medium (Difco).
  • the pH of the medium is preferably 6.0 to 9.0, more preferably pH 7.0.
  • the culture temperature is preferably 20 to 50 ° C, more preferably 45 ° C. Under aerobic conditions, it can be cultured in static culture, shaking culture or rotary culture.
  • ⁇ -amylase can be obtained by purification from the culture or cultured cells containing ⁇ -amylase in the purification step.
  • the method for purifying the enzyme is not particularly limited as long as ⁇ -amylase can be obtained from the culture or cultured cells, but for example, after the culture supernatant is subjected to centrifugation or filter filtration to remove the cells and solids.
  • This enzyme can be purified by combining filtration or concentration with an ultrafiltration filter, salting out, chromatography with a column, adsorption with activated charcoal, and the like.
  • the column to be used is not particularly limited as long as it can be separated from other proteins and impurities without inactivating the enzyme, but it is not particularly limited, but it is an anion exchange chromatography column, a cation exchange chromatography column, and a hydrophobic interaction.
  • examples thereof include a column for chromatography, a column for gel filtration chromatography, and an affinity chromatography column.
  • a TOYOPEARL Butyl-650M column for hydrophobic interaction chromatography and a TOYOPEARL DEAE-650M column for anion exchange chromatography are used.
  • the crude purification or detection of the purified protein can be carried out by a method without particular limitation as long as the protein contained in the sample can be separated and detected.
  • the buffer solution used for enzyme activity measurement, enzyme purification, etc., which will be described later, can be appropriately changed and used according to the purpose of the experiment.
  • the buffer solution preferably has properties that do not affect the activity or stability of the enzyme.
  • Tris-HCl can be used as the buffer solution.
  • the method for producing ⁇ -amylase of the present invention further includes a step of extracting ⁇ -amylase from the cultured bacterial cell.
  • the method for extracting ⁇ -amylase from cultured cells is not particularly limited, and may be any method known to those skilled in the art.
  • ⁇ -amylase can be obtained by, for example, destroying a part or the whole of a cell surface structure (for example, a cell membrane) by solubilizing cells (ultrasonic disruption).
  • the bacterial cells used for extraction may be live or dead. Since the ⁇ -amylase of the present invention produces only maltose from starch, the method for producing ⁇ -amylase of the present invention facilitates the production of maltose.
  • the present invention also provides a method for producing maltose, which comprises treating starch with the ⁇ -amylase of the present invention. Since the enzyme treatment of starch is generally performed on starch gelatinized by heating raw starch, the step of saccharification is performed before the enzyme treatment. The heating temperature varies depending on the type of raw starch, but starch derived from wheat or corn has a high gelatinization temperature. At this time, if ⁇ -amylase having low heat resistance is used, the enzyme is inactivated by the heat of the heated starch, so that it is necessary to cool the heat-gelatinized starch to a temperature suitable for the enzyme reaction.
  • the cooling cost of the heated starch is incurred during the saccharification treatment of starch, and the cooling time is also incurred.
  • the cooling cost of starch can be reduced and / or the cooling time can be shortened, so that it can be suitably used for saccharification treatment of starch. Due to the high heat resistance of this ⁇ -amylase, it can be added to starch after heat treatment, or even to starch during heat treatment.
  • the temperature and pH after the addition of ⁇ -amylase are not particularly limited as long as the enzyme can decompose starch as described in the above enzyme properties, but the ⁇ -amylase of the present invention has a starch temperature of 70 ° C. or lower as a substrate. It can be added at the temperature of 60 ° C or higher and 70 ° C or lower.
  • Starch which is the substrate of the enzyme, can be used for example, soybeans, red beans, barley, wheat, corn, rice, potatoes, sweet potatoes, kudzu, warabi, mung beans, soramame, lotus root, cassava, sago palm, katakuri, etc. Can be used.
  • the enzyme reaction can be stopped by inactivating the enzyme by chemical treatment such as reheating or pH adjustment.
  • Maltose can be produced from starch by the ⁇ -amylase of the present invention. Since the ⁇ -amylase of the present invention produces only maltose from starch, the present invention facilitates the production of maltose.
  • the present invention also provides a method for producing maltose, which comprises treating starch with the microorganism of the present invention.
  • ⁇ -amylase is released from the cells as an extracellular enzyme into the culture medium. Therefore, when the No. 58 strain is cultured with starch, ⁇ -amylase is released to the outside of the cells, and the released ⁇ -amylase reacts with starch to form maltose.
  • the cells to be cultured are not limited to the No. 58 strain, and a recombinant microorganism having the No. 58 strain of ⁇ -amylase may be used.
  • the culture conditions at this time are not particularly limited as long as the microorganisms to be cultured can be cultured, and substances other than starch necessary for growth may be contained. Even if the microorganism does not release ⁇ -amylase to the outside of the cell, if starch is taken into the cell, starch is converted to maltose by ⁇ -amylase in the cell, and maltose is released to the outside of the cell.
  • the recombinant microorganism used is not limited to a microorganism capable of releasing ⁇ -amylase as an extracellular enzyme, but a microorganism in which ⁇ -amylase is released outside the cell is preferable.
  • Maltose can be produced from starch by the microorganism having ⁇ -amylase of the present invention. Since the microorganism having ⁇ -amylase of the present invention produces only maltose from starch, the production of maltose becomes easy.
  • the present invention also provides a method for producing maltose, which comprises treating raw starch with the ⁇ -amylase of the present invention.
  • Conventional ⁇ -amylase has low activity on raw starch and is not suitable for using raw starch as a substrate as it is. Since the ⁇ -amylase of the present invention has high enzymatic activity on raw starch, it can be suitably used for enzyme treatment of raw starch.
  • the state of the raw starch is not particularly limited as long as it can react with the enzyme, but it is preferably in a state of being suspended in an aqueous solution.
  • the temperature and pH after the addition of ⁇ -amylase are not particularly limited as long as the enzyme can decompose starch as described in the above enzyme properties.
  • Starch which is the substrate of the enzyme, can be used for example, soybeans, red beans, barley, wheat, corn, rice, potatoes, sweet potatoes, kudzu, warabi, mung beans, soramame, lotus root, cassava, sago palm, katakuri, etc. Can be used.
  • the enzyme reaction can be stopped by inactivating the enzyme by chemical treatment such as heating or pH adjustment.
  • the ⁇ -amylase of the present invention can produce maltose from raw starch.
  • the present invention also provides a method for modifying a food using the ⁇ -amylase of the present invention or the microorganism of the present invention.
  • Food reforming refers to changing the properties of food. In the present invention, it refers to making the property of a food containing a polysaccharide having an ⁇ -1,4 glucoside bond such as starch into a preferable form.
  • a polysaccharide having an ⁇ -1,4 glucoside bond such as starch
  • the enzyme of the present invention into bread dough before baking, the texture of bread dough is improved, the bread volume is increased, the aging of bread products is prevented (maintenance of softness), and the baking color of bread dough stored frozen or refrigerated. There are measures such as prevention of redness and maintenance of softness of mochi.
  • the food that can be used is not particularly limited, but is preferably a food containing a polysaccharide having an ⁇ -1,4 glucoside bond such as starch.
  • a polysaccharide having an ⁇ -1,4 glucoside bond such as starch.
  • dough such as bread, rice cake, donut, pie, pizza, and bun.
  • the raw materials are not limited to wheat-derived raw materials, but soybeans, red beans, barley, corn, rice (rice), potatoes, sweet potatoes, kudzu, warabi, mung beans, soramame, lotus root, cassava, sago palm, katakuri, etc. can be used. ..
  • the ⁇ -amylase according to the present invention has heat resistance and activity on raw starch, it can be preferably used for food modification operations.
  • strain No. 58 since strain No. 58 has only ⁇ -amylase as an extracellular enzyme, it does not cause excessive starch decomposition or Maillard reaction that occurs when ⁇ -amylase or glucoamylase is used for bread dough. Therefore, strain No. 58 is extremely useful as a source of purified ⁇ -amylase used for these strains.
  • the product is only maltose, it has an advantage that the finished food can have a mellow taste.
  • the heat resistance of this enzyme also has the advantage that the reaction of the substrate can be easily controlled.
  • the optimum pH for this enzyme is neutral 7 Due to its proximity, a pH regulator may not be needed. Since the activity of this enzyme on raw starch can act on ungelatinized starch, it can be used in a wide range of starch products and substrates containing starch.
  • this enzyme has excellent heat resistance, it does not have enzyme activity even at a high temperature of 80 ° C or higher, and the enzyme activity is lost at a high temperature of 80 ° C or higher. Due to this property, when the enzyme is kneaded into bread dough or the like and baked, the enzyme activity can be lost in the baking process. This makes it easy to make preparations such that the enzyme is reacted with the substrate only for the time when the enzyme is desired to react, such as from the beginning to the middle of firing, and finally the enzyme activity is lost.
  • the present invention is also provided in the form of ⁇ -amylase of the present invention or an enzyme preparation containing the microorganism of the present invention.
  • the present enzyme in the enzyme preparation may be a purified enzyme or a crude enzyme stage such as a culture supernatant.
  • the enzyme preparation may be composed of ⁇ -amylase alone, or may be appropriately blended with additives as long as the enzymatic properties are not significantly impaired. Examples of the form of the enzyme preparation include tablets, pills, powders, granules, capsules, and liquids.
  • the enzyme preparation may be encapsulated in a sprayer such as a spray, and may contain an enzyme in addition to amylase.
  • Examples of the enzyme contained together with this enzyme include debranching enzyme, isoamylase, pullulanase, glucosidase, glucoamylase, ⁇ -amylase, protease, lipase, phosphatase, xylanase, etc., and even if one or more of these enzymes are used in combination. Good.
  • Additives include, for example, binders (rubber arabic, gelatin, sorbitol, tragant, polyvinylpyrrolidone, etc.), fillers (lactose, sugar, calcium phosphate, sorbitol, glycine, etc.), disintegrants (crystalline cellulose, etc.), non-aqueous additives.
  • binders rubber arabic, gelatin, sorbitol, tragant, polyvinylpyrrolidone, etc.
  • fillers lactose, sugar, calcium phosphate, sorbitol, glycine, etc.
  • disintegrants crystalline cellulose, etc.
  • Shapes (almond oil, fractionated coconut oil or oily esters such as glycerin, propylene glycol, polyethylene glycol, ethyl alcohol, etc.), preservatives (methyl or propyl p-hydroxybenzoate, sorbitol, tocopherol, etc.), pH adjustment Agents (sodium hydrogen carbonate, potassium carbonate, citrate, acetate, etc.), thickeners (methylcellulose, etc.), antioxidants (vitamin C, vitamin E, etc.), fragrances (synthetic fragrances, natural fragrances, esters, etc.) , Emulsifiers (lecithin, sorbitol monooleate, sucrose fatty acid esters, etc.), swelling agents (ammonium carbonate, etc.), suspending agents (syrup, gelatin, water-added edible fat, etc.) and the like.
  • pH adjustment Agents sodium hydrogen carbonate, potassium carbonate, citrate, acetate, etc.
  • thickeners methylcellulose, etc.
  • This enzyme preparation can be widely used in applications such as starch and other polysaccharides having an ⁇ -1,4 glucosidic bond decomposed in maltose units to produce maltose, and in food modification applications. For example, adding an enzyme agent to dough such as bread, rice cake, donut, pie, pizza, and bun. In addition, it is added to grains such as wheat, soybeans, red beans, barley, corn, rice (rice), potatoes, sweet potatoes, kudzu, warabi, mung beans, soramame, lotus roots, cassava, sago palm, and katakuri that have starch, and their crushed products. can do. Pretreatment such as crushing, heating, or chemical treatment may be performed before the addition of the enzyme preparation. The treatment conditions after the addition of the enzyme preparation can be appropriately determined by those skilled in the art.
  • Experiment 1 Screening and identification of bacteria having thermostable ⁇ -amylase ⁇ -amylase-producing heat-resistant bacteria were isolated and isolated from soil samples.
  • the soil of the screening source was obtained from the soil in Osaka Prefecture.
  • a starch assimilating strain was isolated from this soil.
  • the isolated strain was cultured in a medium for amylase-producing bacteria (Table 1).
  • the basic pH was 7.0, and the medium composition and pH were changed according to the experiment.
  • 1.0% gellan gum was added in addition to the above composition, and the mixture was solidified on a plate before use.
  • the isolated starch assimilating strain was inoculated into a test tube containing 6 ml of a liquid medium for amylase-producing bacteria, and cultured at 60 ° C. for 3 days with shaking. After completion of the culture, the culture supernatant was collected by centrifuging the culture solution (3200 rpm, 10 minutes, 4 ° C), and this was used as a crude enzyme sample. To 100 ⁇ l of this crude enzyme sample, 100 ⁇ l of a substrate solution (1.5% soluble starch (manufactured by Merck & Co., Inc.), 100 mM pH7.0Tris-HCl, 5 mM CaCl 2 ) was added, and the mixture was reacted overnight at 37 ° C. After the reaction, 5 ⁇ l of the reaction solution was subjected to thin layer chromatography.
  • a substrate solution (1.5% soluble starch (manufactured by Merck & Co., Inc.)
  • 100 mM pH7.0Tris-HCl 5 mM Ca
  • TLC Thin Layer Chromatography
  • Silica gel 60 F254 (manufactured by Merck & Co., Inc.) was used as the silica gel plate used for thin layer chromatography.
  • the developing solvent used was 1-butanol / ethanol / chloroform / 25% (w / w) ammonia solution: (4/5/2/8, v / v / v / v).
  • Maltose was detected by spraying the detection solution (concentrated sulfuric acid containing 1% vanillin) on the silica gel plate after development three times and heating at 120 ° C. for several minutes to develop color.
  • Amylase activity measurement 100 ⁇ l of an enzyme sample (culture supernatant or crude or purified enzyme described below) was placed in a test tube and pre-incubated at 37 ° C. for 10 minutes. 100 ⁇ l of substrate solution (1.5% soluble starch, 100 mM Tris-HCl, 5 mM CaCl 2 , pH 7.0, 37 ° C) was added and reacted at 37 ° C for 10 minutes. The enzyme reaction was stopped by adding 400 ⁇ l of DNS solution (0.5% dinitrosalicylic acid, 0.4N NaOH, 30% potassium sodium tartrate tetrahydrate). Then, the mixed solution was boiled at 100 ° C.
  • substrate solution 1.5% soluble starch, 100 mM Tris-HCl, 5 mM CaCl 2 , pH 7.0, 37 ° C
  • the enzyme reaction was stopped by adding 400 ⁇ l of DNS solution (0.5% dinitrosalicylic acid, 0.4N NaOH, 30% potassium sodium tartrate tetrahydrate). Then, the
  • the temperature stability of the amylase of the acquired No. 58 strain was evaluated.
  • the obtained No. 58 strain was cultured, and the culture supernatant was collected and used as a culture supernatant sample.
  • a moderately diluted culture supernatant sample was placed in a microtube, kept warm at 60 ° C., sampled after 10, 20, 40, and 80 minutes, and cooled on ice.
  • the residual enzyme activity of the sample after cooling was measured by quantifying the amount of reducing sugar produced, and the temperature stability of the obtained amylase at 60 ° C. was evaluated.
  • TMK-672 strain (TBA strain) was treated in the same manner, and the obtained sample was used. The result is shown in FIG. The data is shown as a relative value with the value of 0 minutes of processing time as 100%. No. 58 in FIG. 2 represents the strain of the present invention, and TBA represents the comparison target. Furthermore, it was confirmed whether the obtained amylase was ⁇ -amylase by determining the relationship between the amount of reducing sugar produced and the relative KI / I 2 value.
  • the relative KI / I 2 value was calculated as follows. Take 200 ⁇ l of the enzyme solution in a test tube, pre-incubate at 37 ° C for 10 minutes, add 400 ⁇ l of the same pre-incubated substrate solution, react at 37 ° C for 10 minutes, and 1 ml of the reaction terminator (0.5N acetic acid and 0.5N). The reaction was stopped by adding (a mixture of HCl in a ratio of 5: 1). After the reaction was stopped, 200 ⁇ l of the reaction solution was placed in another test tube, 5 ml of iodine solution (0.005% I 2 , 0.05% KI) was added, and after 20 minutes, absorption at 660 nm was measured with a spectrophotometer.
  • the relative KI / I 2 value when the amount of reducing sugar was 0 was set to 100%.
  • the concentration of the sample to be measured was appropriately changed, and this value was plotted against the value obtained by the above-mentioned reducing sugar quantification method.
  • ⁇ -amylase derived from human saliva as a positive control of ⁇ -amylase
  • ⁇ -amylase derived from TMK-672 strain as a positive control of ⁇ -amylase
  • the pH was adjusted to 7.0.
  • ampicillin with a composition of 100 ⁇ g / ml was added to the medium.
  • a 10% SDS solution was added to the Solution I solution containing the cultured cells so that the final concentration was 0.5%, and the mixture was shaken for 10 minutes. Further, a 5M NaCl solution was added to this mixed solution so that the final concentration of NaCl was 0.7M or more, and the mixture was shaken for 10 minutes. Then, phenol / chloroform extraction was performed, and the obtained phenol / chloroform-added solution was centrifuged (13,500 rpm, 10 minutes, 4 ° C.) to separate into an aqueous layer and an oil layer, and the aqueous layer was recovered.
  • the 16S rRNA gene was amplified by PCR.
  • the template is the extracted chromosomal DNA, and the primer is the forward primer (5'-AGRTTTGATYHTGGYTCAG 3': SEQ ID NO: 3) designed based on the highly conserved region of the bacterial 16S rRNA gene.
  • a reverse primer (5'-TGACGGGCGGTGTGTACAAG -3': SEQ ID NO: 4) was used.
  • the 16S rRNA region was amplified by PCR using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio), and the temperature cycle was 98 ° C for 10 seconds, 55 ° C for 5 seconds, and 72 ° C for 90 seconds for 30 cycles.
  • the obtained PCR product was inserted into pBluescript II SK (+) (manufactured by Stratagene) digested with EcoRV, transformed into Escherichia coli, and then the plasmid was extracted. Sequence analysis was outsourced to Eurofins Genomics. GENETY X-WIN Ver. 7 (Genetics) was used for the base sequence analysis, and NCBI (http://www.ncbi.nlm.nih.gov/) BLAST search was used for the base sequence identity search.
  • the No. 58 strain of the present invention was identified as the Bacillus halosaccharovorans No. 58 strain (No. 58 strain). This strain was deposited on April 12, 2019 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (Room 122, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture). The accession number is NITE BP-02937.
  • the adsorbent was eluted with a reverse linear gradient (1.0 L) of 20 mM Tris-HCl buffer (pH 7.0) containing 20-0% saturated ammonium sulfate, and fractionated by 10 ml. The activity of each fraction was measured, the fraction showing ⁇ -amylase activity was recovered, ammonium sulfate was added and dissolved to a saturation concentration of 80%, and a precipitate was formed at 4 ° C. overnight. The precipitate was recovered by centrifuging this (10,800 rpm, 30 minutes, 4 ° C).
  • the recovered precipitate was dissolved in a small amount of 20 mM Tris HCl buffer (pH 7.0) and dialyzed against a large excess of 20 mM Tris-HCl buffer (pH 7.0) using a dialysis membrane.
  • Fig. 4 shows the results of subjecting the purified enzyme obtained by a series of purification experiments to SDS-PAGE. From FIG. 4, a single band showing a molecular weight of 59.4 kDa was obtained by purification, indicating that this enzyme could be purified as a single enzyme. After that, the obtained purified enzyme was used.
  • Experiment 3 ⁇ -amylase sequence analysis N-terminal amino acid sequence analysis After performing SDS-PAGE using a purified sample, the gel was placed in blotting solution C (0.02% SDS, 25 mM Tris, 20% methanol, 25 mM boric acid) 5 Shake gently for minutes. Prepare 6 sheets of filter paper cut into gel size, and blotting solution A (0.02% SDS, 300mM Tris, 20% methanol), blotting solution B (0.02% SDS, 25mM Tris, 20% methanol), and blotting solution C, 2 each. Soaked sheet by sheet and shaken for 15 minutes.
  • blotting solution C 0.02% SDS, 25 mM Tris, 20% methanol
  • a PVDF membrane manufactured by MILLIPORE
  • MILLIPORE a PVDF membrane
  • the PVDF film after transfer was lightly washed with ultrapure water, stained with Ponceau S stain (0.1% Ponceau S, 5% acetic acid) for 5 minutes, and then decolorized with ultrapure water. A part of the target band was cut out, collected in a tube, and dried. Sequence analysis was outsourced to the Hokkaido University Global Facility Center Equipment Analysis Contract Service. As a result of analyzing the N-terminal amino acid sequence, it was found to be H 2 N-Glu-Ile-Lys-Thr-Asp-Tyr-Lys-Ala-Ser-Val-.
  • the ⁇ -amylase gene was amplified by PCR.
  • the template is the No. 58 strain chromosomal DNA prepared by the method described in Experiment 1, and the primer is a forward primer designed based on a highly conserved region in bacterial ⁇ -amylase (5'-GTNTGGTGGGGNTAYGTNGA-3). ': SEQ ID NO: 5) and a reverse primer (5'-GGRTTRTTCATYTGCCARTG-3': SEQ ID NO: 6) were used.
  • PCR amplification was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.), and the temperature cycle was 98 ° C for 10 seconds, 55 ° C for 5 seconds, and 72 ° C for 50 seconds for 30 cycles.
  • the obtained PCR product was ligated to pBluescript II SK (-) digested with the restriction enzyme EcoRV and transformed into Escherichia coli.
  • the cells were inoculated into 2 ml of 2 ⁇ TY medium (added 100 ⁇ g / ml ampicillin) and cultured with shaking at 30 ° C. for 24 hours.
  • a plasmid was extracted from the transformed Escherichia coli and sequence analysis was performed to confirm that the plasmid contained the ⁇ -amylase sequence.
  • Primer PS-F (5'-GGCAGCCCAGGTAATTTCTAC-3': SEQ ID NO: 7) and PS-R (5'-GGAGGGTTGACTTCACTCCA-3') designed from the determined ⁇ -amylase gene sequence using the plasmid containing the obtained gene as a template.
  • a DIG (digoxigenin) -labeled probe was synthesized using SEQ ID NO: 8). No.
  • 58 strain genomic DNA was obtained from 10 types of restriction enzymes (Sac I, BamHI, Xba I, Sal I, Sph I, Hind III, Bgl II, Spe I, Nhe I, Xho I). EcoR I and Kpn I, which have cleavage sites, were digested overnight with a combination of each, and Southern blot analysis was performed using the synthesized probe. From this, it was predicted that the Bgl II digestion gene fragment of about 6.3 kb contained the entire length of the target gene. Therefore, the Bgl II digestion gene fragment corresponding to the size of about 6.3 kb was recovered and used with a BamHI restriction enzyme.
  • the plasmid containing the gene of interest was obtained by screening by the PCR method using the primers used when preparing the probe.
  • the obtained plasmid was digested with various restriction enzymes, subcloned into pBluescript II SK (-), and then sequence analysis was performed.
  • the result of sequence analysis is shown in FIG.
  • the resulting sequence consists of 2536 bp (SEQ ID NO: 11), of which the gene is composed of an open reading frame (ORF) (SEQ ID NO: 1) consisting of 1,659 bp, with a ribosome binding site (RBS) 6 bp upstream of the start codon. It was found that there is a promoter region consisting of a -10 region near 56 bp upstream and a -35 region near 82 bp upstream.
  • ORF open reading frame
  • RBS ribosome binding site
  • this gene was synthesized as a precursor protein consisting of 552 amino acids (SEQ ID NO: 12, the corresponding base sequence is SEQ ID NO: 1).
  • SEQ ID NO: 12 the corresponding base sequence is SEQ ID NO: 1.
  • a signal sequence consisting of 29 amino acids (indicated by the underline in the figure) exists from this sequence, and the mature protein is composed of 523 amino acids and has an amino acid sequence.
  • the molecular weight calculated from the above was 59,403 (SEQ ID NO: 2, the corresponding base sequence is SEQ ID NO: 13).
  • the mature protein is composed of a catalytic domain classified into Glycoside hydrolase family 14 (GH14) in the N-terminal region and a substrate binding domain classified into Carbonate-binding module 20 (CBM20) in the C-terminal side. It was.
  • GH14 Glycoside hydrolase family 14
  • CBM20 Carbonate-binding module 20
  • the enzyme solution was mixed with a 50 mM universal buffer of pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.2, 8.5, 9.0, 9.5, 10.0, 10.75.
  • each mixed solution was allowed to stand at 4 ° C. for 16 hours.
  • the pH was returned to 7.0 by diluting this mixed solution 10-fold with 100 mM Tris-HCl buffer (pH 7.0).
  • the residual activity of these diluted mixed solutions was measured by reacting these diluted mixed solutions at 37 ° C. for 10 minutes in the same manner as in the above-mentioned amylase activity measurement.
  • the measurement result of pH optimum is shown in FIG.
  • FIGS. 6 and 7 the vertical axis is the relative activity value with the measured maximum activity value as 100%. From FIG. 6, this enzyme showed the maximum activity at around pH 7.0. From FIG. 7, it was clarified that this enzyme shows a residual activity of 80% or more in the pH range of 5.0 to 9.0.
  • Optimal temperature and temperature stability In order to determine the optimum temperature for ⁇ -amylase, the enzyme solution was reacted at each temperature of 37 to 70 ° C. for 10 minutes in the same manner as the above amylase activity measurement except for the temperature. The enzyme activity was measured.
  • the temperature stability is the same as the above amylase activity measurement by treating the enzyme solution (20 mM Tris-HCl buffer, pH 7.0) at 37, 45, 50, 55, 60, 65, 70 ° C. for 30 minutes and then cooling. The residual activity was measured by reacting at 37 ° C. for 10 minutes by the above method.
  • FIG. 8 shows the measurement results of the optimum temperature
  • FIG. 9 shows the residual activity of each ⁇ -amylase at 60 ° C.
  • FIG. 10 shows the residual activity of each ⁇ -amylase at 65 ° C.
  • FIG. 8 shows the relative activity value with the measured maximum activity value as 100% for each temperature.
  • the value of 0 minutes of the processing time is set as 100% and shown as relative values. From FIG. 8, it was found that the optimum temperature of this enzyme was around 50 ° C. From FIG. 9, the activity of ⁇ -amylase derived from barley, ⁇ -amylase derived from Bacillus cereus, and ⁇ -amylase derived from Brevibacillus sp. TMK-672 strain decreased in this order, and 60 to 60 to soybean-derived ⁇ -amylase known to have heat resistance. After 80 minutes of treatment, the residual activity was about 80%. On the other hand, ⁇ -amylase derived from strain No.
  • Raw starch degrading activity An experiment was conducted to compare the degrading activity of ⁇ -amylase on raw starch. As the raw starch, those derived from corn and those derived from wheat were used. Each raw starch 1.0 mg / ml was reacted in the presence of 20 mM Tris-HCl buffer (pH 7.0), 5 mM CaCl 2 , and enzyme 0.1 ⁇ g / ml, and an appropriate amount of reaction solution was added to a test tube containing 0.4 ml DNS solution. The reaction was stopped by adding to, and the raw starch degrading activity was evaluated by the method for quantifying reduced sugars described in Experiment 1.
  • the ⁇ -amylase of the present invention As the ⁇ -amylase, the ⁇ -amylase of the present invention, the ⁇ -amylase derived from barley, and the ⁇ -amylase derived from soybean were used.
  • Barley-derived ⁇ -amylase is known to have the activity of decomposing raw starch
  • soybean-derived ⁇ -amylase is known to have weak raw starch activity but high heat resistance.
  • Wheat-derived starch was reacted at 60 ° C
  • corn-derived starch was reacted at 70 ° C.
  • the reaction results for wheat-derived starch are shown in FIG. 11, and the reaction results for corn-derived starch are shown in FIG. From FIG.
  • the cells were collected by centrifugation (10,000 rpm, 2 minutes, 4 ° C), suspended in 1 ml of 20 mM Tris-HCl buffer pH 7.0, 5 mM CaCl 2 , and then used with Handy sonic disruptor UD1 (manufactured by Tomy Seiko). , 30 seconds x 4 cycles of ultrasonic crushing.
  • the amylase activity was measured using the supernatant obtained by centrifuging the crushed solution (15,000 rpm, 10 minutes, 4 ° C) as a crude enzyme, and the production of reducing sugars was confirmed using soluble starch as a substrate.
  • the reaction product was detected using TLC, it was only maltose.
  • the enzyme activity was 55.1 U / ml per 1 ml of culture medium.
  • Site-specific mutagenesis into ⁇ -amylase gene Site-specific mutagenesis was performed into the ⁇ -amylase gene derived from strain No. 58 to prepare a mutant enzyme, and its enzyme activity was measured. Site-specific mutagenesis into the ⁇ -amylase gene was performed by the PCR-mega primer method. First, using a plasmid containing the coding region of the ⁇ -amylase gene and the ribosome binding site as a template, four types of mutagenesis primers (SEQ ID NOs: 14 to 17) and exp-R primers (SEQ ID NOs: 10) shown in Table 4 below were used.
  • PCR was performed in the same manner as in "cloning the ⁇ -amylase gene” to obtain a mega primer.
  • RBS-F primer SEQ ID NO: 9
  • a plasmid containing the coding region of the ⁇ -amylase gene and the ribosome binding site as a template PCR was performed in the same manner to generate mutations.
  • the full length of the introduced ⁇ -amylase gene was obtained.
  • An expression plasmid was constructed from this gene fragment using the same method as in Experiment 5, and the transformant was transformed into the E. coli BL21 (DE3) strain to obtain a transformant producing ⁇ -amylase mutant enzyme. ..
  • Each of the obtained transformants was cultured using the same method as in Experiment 5, and the cells were disrupted to obtain a crude enzyme.
  • the crude enzyme was purified using the same method as in Experiment 2.
  • the purification result of each mutant enzyme is shown in FIG.
  • the temperature stability of these purified enzymes was evaluated in the same manner as in "Confirmation of ⁇ -amylase”.
  • Each enzyme sample was placed in a microtube, kept warm at 65 ° C., sampled after 15, 30 and 60 minutes, and cooled on ice.
  • the residual enzyme activity of the sample after cooling was measured by quantifying the amount of reducing sugar produced, and the temperature stability of the obtained amylase at 65 ° C. was evaluated.
  • an unmutated purified enzyme was also treated in the same manner to evaluate the temperature stability.
  • the result is shown in FIG.
  • the data is shown as a relative value with the value of 0 minutes of processing time as 100%.
  • wt represents ⁇ -amylase of the No. 58 strain of the present invention
  • T4P, T15K, T15R, and Q306D each represent a mutant enzyme (for example, in the case of T4P, the fourth amino acid residue T of SEQ ID NO: 2 is P. Indicates that it is mutated).
  • the residual activity after 60 minutes was 37.7% for ⁇ -amylase before mutation, 80.9% for T4P, 50.1% for Q306DE, 44.4% for T15K, and 44.2% for T15R. It was revealed that the thermostability was improved as compared with the ⁇ -amylase before the mutation.
  • the ⁇ -amylase mutant enzymes contained in the obtained four transformants are mutations in which threonine at amino acid number 4 of the amino acid sequence shown in SEQ ID NO: 2 is converted to proline (SEQ ID NO: 18: T4P in FIG. 14). , Or a mutation in which threonine at amino acid number 15 is converted to lysine (SEQ ID NO: 19: T15K in FIG.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Polymers & Plastics (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/JP2020/016434 2019-04-15 2020-04-14 新規βアミラーゼ及びその利用・製造法 Ceased WO2020213604A1 (ja)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2021514172A JP7649547B2 (ja) 2019-04-15 2020-04-14 新規βアミラーゼ及びその利用・製造法
US17/603,836 US12473542B2 (en) 2019-04-15 2020-04-14 β-amylase and method for utilization and production thereof
CN202080028731.0A CN114072519B (zh) 2019-04-15 2020-04-14 新型β淀粉酶及其应用和制备方法

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2019-077041 2019-04-15
JP2019077041 2019-04-15
JP2019-145697 2019-08-07
JP2019145697 2019-08-07

Publications (1)

Publication Number Publication Date
WO2020213604A1 true WO2020213604A1 (ja) 2020-10-22

Family

ID=72837788

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2020/016434 Ceased WO2020213604A1 (ja) 2019-04-15 2020-04-14 新規βアミラーゼ及びその利用・製造法

Country Status (4)

Country Link
US (1) US12473542B2 (https=)
JP (1) JP7649547B2 (https=)
CN (1) CN114072519B (https=)
WO (1) WO2020213604A1 (https=)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4867487A (https=) * 1971-12-17 1973-09-14
JPS61282073A (ja) * 1985-06-07 1986-12-12 Nakano Vinegar Co Ltd 新規アミラ−ゼ，該アミラ−ゼをコ−ドする遺伝子を含む組換えプラスミド，該プラスミドにより形質転換された微生物および該微生物による新規アミラ−ゼの製造法
JPS62201577A (ja) * 1986-02-19 1987-09-05 ノボ　ノルディスク　アクティーゼルスカブ β−アミラ−ゼ酵素
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 (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6279746A (ja) 1985-10-01 1987-04-13 Showa Sangyo Kk でんぷん質食品の老化を防止する方法
DK2454942T3 (en) 2009-07-17 2018-07-30 Amano Enzyme Inc PROCEDURE FOR IMPROVING FOODS USING BETA AMYLASE
CN107058263B (zh) * 2017-01-26 2020-11-06 福建福大百特生物科技有限公司 一种新型β-淀粉酶的高效制备方法
CN107254459A (zh) * 2017-06-29 2017-10-17 中国科学院青岛生物能源与过程研究所 耐热β‑淀粉酶‑海藻糖合成酶融合酶、其表达基因以及分泌该融合酶的工程菌与应用
CN107164345B (zh) 2017-07-06 2019-09-03 江南大学 一种热稳定性提高的β-淀粉酶突变体

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4867487A (https=) * 1971-12-17 1973-09-14
JPS61282073A (ja) * 1985-06-07 1986-12-12 Nakano Vinegar Co Ltd 新規アミラ−ゼ，該アミラ−ゼをコ−ドする遺伝子を含む組換えプラスミド，該プラスミドにより形質転換された微生物および該微生物による新規アミラ−ゼの製造法
JPS62201577A (ja) * 1986-02-19 1987-09-05 ノボ　ノルディスク　アクティーゼルスカブ β−アミラ−ゼ酵素
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

Also Published As

Publication number Publication date
US12473542B2 (en) 2025-11-18
JP7649547B2 (ja) 2025-03-21
US20230077057A1 (en) 2023-03-09
CN114072519B (zh) 2025-03-04
CN114072519A (zh) 2022-02-18
JPWO2020213604A1 (https=) 2020-10-22

Similar Documents

Publication Publication Date Title
JP4550587B2 (ja) 熱安定性α−アミラーゼ
KR100319442B1 (ko) 플루라나아제, 이를 제조하는 미생물, 이플루라나아제의 제조방법과 이의 용도
CN102595910B (zh) 利用β-淀粉酶的食品改性方法
ES2565843T3 (es) Beta-amilasa, gen que la codifica y procedimiento de preparación de la misma
KR20120103545A (ko) 말토트리오실 전이효소, 그 제조 방법 및 용도
CN106460023A (zh) 使用α‑葡糖苷酶来酶水解二糖和低聚糖
CN109385413B (zh) 葡萄糖淀粉酶TlGA1931及其基因和应用
JP2024091998A (ja) マルトトリオース生成アミラーゼ
WO2016126294A1 (en) Truncated pullulanases, methods of production, and methods of use thereof
CN101657544A (zh) 新型α-半乳糖苷酶
JP7649547B2 (ja) 新規βアミラーゼ及びその利用・製造法
US20210403957A1 (en) Application of trehalase in fermentative production
KR101706451B1 (ko) 마이크로불비퍼 속 유래 말토트리오스 생성용 아밀라아제 및 이를 이용한 말토트리오스 생산방법
AU2017222025A1 (en) Alpha amylase variant and use thereof
CN107312764B (zh) α淀粉酶变体
JP2009207452A (ja) Ｂａｃｉｌｌｕｓｃｏａｇｕｌａｎｓに属する菌株を用いたメリビオースの製造方法
BE1007313A3 (fr) Pullulanase, microorganismes la produisant, procedes de preparation de cette pullulanase et utilisations de celle-ci.
BE1007723A6 (fr) Pullulanase, microorganismes la produisant, procedes de preparation de cette pullulanase et utilisations de celle-ci.
KR101655237B1 (ko) 고정화된 아밀레이즈를 이용한 말토덱스트린의 연속 제조방법
PL211354B1 (pl) Nowy szczep bakterii Bacillus subtilis i sposób otrzymywania α-amylazy przy użyciu nowego szczepu
KR20010037359A (ko) Ｌ62 리파제를 생산하는 신균주 스타필로코커스 헤모리티커스 Ｌ62(ＫＣＴＣ 8957Ｐ) 및 형질전환된 대장균 ＢＬ21(ＤＥ3)/ｐＳＨＭＬ(ＫＣＴＣ 8956Ｐ)을 이용한 Ｌ62 리파제의 대량생산법

Legal Events

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

Ref document number: 20792059

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021514172

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20792059

Country of ref document: EP

Kind code of ref document: A1

WWG Wipo information: grant in national office

Ref document number: 202080028731.0

Country of ref document: CN

WWG Wipo information: grant in national office

Ref document number: 17603836

Country of ref document: US