WO2013168438A1 - Protéine ayant une activité lactase, gène codant pour ladite protéine, vecteur recombinant portant ledit gène, transformant, procédé de fabrication de ladite protéine, et utilisation de ladite protéine - Google Patents

Protéine ayant une activité lactase, gène codant pour ladite protéine, vecteur recombinant portant ledit gène, transformant, procédé de fabrication de ladite protéine, et utilisation de ladite protéine Download PDF

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WO2013168438A1
WO2013168438A1 PCT/JP2013/050559 JP2013050559W WO2013168438A1 WO 2013168438 A1 WO2013168438 A1 WO 2013168438A1 JP 2013050559 W JP2013050559 W JP 2013050559W WO 2013168438 A1 WO2013168438 A1 WO 2013168438A1
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protein
seq
lactase activity
dna
gene
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PCT/JP2013/050559
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English (en)
Japanese (ja)
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公安 礒部
芳彦 廣瀬
貴史 小山
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国立大学法人岩手大学
天野エンザイム株式会社
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Priority to JP2014514391A priority Critical patent/JP6096179B2/ja
Publication of WO2013168438A1 publication Critical patent/WO2013168438A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—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)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • C12N9/2471—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/195—Proteins from microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • 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)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a protein having lactase activity. More specifically, an acid-resistant protein having lactase activity, a gene encoding the protein, a recombinant vector containing the gene, a transformant, an enzyme preparation using the protein, a pharmaceutical composition, a quasi-drug composition, The present invention also relates to a food composition and a method for producing an acid-resistant protein having lactase activity.
  • Lactase is a kind of ⁇ -galactosidase and is an enzyme that hydrolyzes lactose (lactose) into glucose and galactose. Industrially, it is also used to produce low-lactose milk, ice cream, and other syrups from whey. In humans, it is present in a large amount in epithelial cells of the small intestine and functions as a digestive enzyme for breaking down lactose contained in dairy products into glucose and galactose and absorbing them in the small intestine.
  • Lactose is a disaccharide and cannot be absorbed as it is, but can only be absorbed in the small intestine by being decomposed into glucose and galactose by lactase.
  • lactase decreases in activity at the weaning stage in mammals. Even in humans, the activity is high in the perinatal period, but the activity naturally decreases around the age of 5. In general, as you finish the lactation period and become an adult from an infant, you will not consume large amounts of milk containing lactose, so even if you lose a lactase activity, There are often no health problems.
  • lactose intolerance Lactose intolerance that occurs after the lactation period is called late-onset lactose intolerance, and although rare, there are cases in which lactase is congenitally deficient. being called.
  • lactose intolerance lactose that was not decomposed in the small intestine is not absorbed in the small intestine and reaches the large intestine as it is. Lactose that reaches the large intestine increases the osmotic pressure in the large intestine. When the osmotic pressure in the large intestine increases, the absorption of water in the large intestine is hindered, resulting in osmotic diarrhea. In addition, lactose that has reached the large intestine is fermented by microorganisms and the like present in the large intestine, and gas such as lactic acid and carbon dioxide is generated, resulting in symptoms such as discomfort, abdominal pain, and diarrhea.
  • lactose in milk is decomposed by lactose-degrading enzyme, and then sugar is converted into alcohol and carbon dioxide by ethanol fermentation, followed by drying to produce powdered milk, thereby obtaining low lactose powdered milk.
  • Patent Document 2 discloses a technique related to Lactobacillus lactic acid bacteria having an ability to improve lactose intolerance, in which both intestinal adhesion and lactose-degrading enzyme activity are enhanced.
  • Patent Document 3 discloses a lactase composition containing ⁇ -galactosidase, an excipient, and less than about 10% reducing sugar, and having improved stability by avoiding the use of a deliquescent excipient. ing.
  • the method of ingesting a lactase preparation is a method in which lactase is administered directly, so that a high effect can be expected.
  • lactase preparations have a problem that their stability under acidic conditions is extremely low.
  • the stomach pH during fasting is 1 to 2, and therefore, when a lactase preparation is ingested during fasting, it is deactivated in the stomach. Therefore, in practice, a method of taking a lactase preparation at the time of eating together with the first mouth or after the first mouth is taken.
  • Non-Patent Document 1 discloses a technique relating to acid-resistant lactase derived from Bisporapsp. And having high stability at pH 1-2.
  • the preparation uses acid-resistant lactase having high stability at the same pH 1-2 as that in the fasting stomach, a method of taking the lactase preparation in advance before meals can be realized.
  • Non-Patent Document 1 As described above, in recent years, a technique relating to acid-resistant lactase having high stability at pH 1 to 2 has been developed. However, although the lactase of Non-Patent Document 1 is certainly stable at pH 1-2, the optimum pH is also on the acidic side, and the activity value at pH 4-5 is only about 30% of the optimum pH (non- (See Patent Document 1 Figure 3. a).
  • Non-Patent Document 1 The stomach pH during fasting is 1-2, but it rises to pH 4-5 by eating. Therefore, when lactose is actually decomposed after a meal, the lactase of Non-Patent Document 1 has a problem that it cannot exhibit its original activity.
  • the main object of the present invention is to provide a novel protein having lactase activity that is stable at pH 1-2 and has high activity at pH 4-5.
  • the present invention first provides a protein having the following physicochemical properties.
  • the protein according to the present invention is stable in the stomach on an empty stomach and after a meal, and also exhibits high activity in the stomach after a meal.
  • the protein according to the present invention is not particularly limited as long as it has the physicochemical properties described above, and (6) Km value: 2-nitrophenyl- ⁇ -D-galactopyranoside It is preferable that it is about 0.19 mM with respect to.
  • the origin of the protein according to the present invention is not particularly limited as long as it is a protein specified by the above physicochemical properties. Examples thereof include those derived from microorganisms belonging to the genus Teratosphaeria. In this case, examples of microorganisms belonging to the genus Teratosphaeria include Teratosphaeria acidotherma.
  • the present invention provides the following protein (a), (b) or (c).
  • A a protein comprising the amino acid sequence represented by SEQ ID NO: 17;
  • B A protein having a lactase activity, comprising an amino acid sequence in which one to several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 17.
  • C A protein having a lactase activity, comprising an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 17.
  • the present invention also provides a gene encoding the protein. As a specific example, the gene consisting of DNA described in the following (a), (b) or (c) can be mentioned.
  • A DNA consisting of the base sequence represented by SEQ ID NO: 16 or 20.
  • a DNA encoding a protein having a lactase activity comprising a base sequence in which one to several bases are deleted, substituted and / or added in the base sequence represented by SEQ ID NO: 16 or 20.
  • C DNA encoding a protein having a lactase activity, comprising a base sequence having 90% or more homology with the base sequence represented by SEQ ID NO: 16 or 20.
  • the protein according to the present invention can be suitably used as an enzyme preparation by containing it as an active ingredient.
  • the protein and enzyme preparation according to the present invention can be suitably used for a pharmaceutical composition, a quasi-drug composition, or a food composition.
  • the method for producing a protein according to the present invention is not particularly limited.
  • a protein having lactase activity is collected from a culture obtained by culturing the microorganism capable of producing the protein or the transformant in a nutrient medium. Can be manufactured.
  • the protein according to the present invention is stable in the stomach on an empty stomach and exhibits high activity in the stomach after a meal, so that an enzyme preparation, a pharmaceutical composition, or a quasi-drug containing this as an active ingredient
  • the composition or the food composition can be ingested before a meal, and can also realize a very high lactose decomposition action even after a meal.
  • the range of options for taking an enzyme preparation or the like in lactose intolerance is widened, and symptoms can be effectively suppressed by lactose intolerance.
  • FIG. 4 is a graph instead of a drawing showing the relative activity (%) with respect to pH in Example 3.
  • FIG. 6 is a drawing-substituting graph showing residual activity (%) relative to pH in Example 4.
  • FIG. 10 is a drawing-substituting graph showing relative activity (%) with respect to temperature in Example 5.
  • FIG. 10 is a drawing-substituting graph showing remaining activity (%) with respect to time in Example 6.
  • FIG. 10 is a drawing-substituting graph showing the residual activity (%) with respect to temperature in Example 6.
  • FIG. 6 is a drawing-substituting photograph showing the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis in Example 7.
  • the protein according to the present invention is a protein having physicochemical properties and lactase activity described below.
  • the method for measuring lactase activity is not particularly limited, and any known method can be freely selected.
  • lactase activity was measured by the method described in Examples described later.
  • lactose acts on lactose (lactose) and hydrolyzes it into glucose and galactose.
  • lactose and lactose are used in the same meaning.
  • Lactose is a kind of disaccharide having a chemical formula of C12H22O11 and a molecular weight of 342.3 in which D-galactose and D-glucose are linked by ⁇ -1,4 galactosides.
  • the protein according to the present invention decomposes lactose into glucose and galactose by hydrolyzing the ⁇ -1,4 galactosidic bond of lactose.
  • the protein according to the present invention has the highest lactase activity at pH around 3.0 to 4.0 under the reaction conditions of 37 ° C. for 15 minutes and 3 hours, and has an activity of 80% or more at the optimum pH at pH 4 to 5. This is shown (see Example 3 described later).
  • lactose lactose
  • the protein according to the present invention exhibits a lactase activity of 40% or more at the optimum pH at pH 1.0 and 7.0 under the reaction conditions of 37 ° C. for 15 minutes and 3 hours (see Example 3 described later). ). Therefore, even if the stomach pH is more acidic or neutral than usual due to the contents of food and drinks or for some reason, it is possible to maintain a certain level of lactose decomposition action.
  • the protein according to the present invention is stable at pH 1.5 to 7 (see Example 4 described later). That is, the protein of the present invention is stable both in the fasting stomach and in the stomach after a meal. Therefore, the enzyme preparation, pharmaceutical composition, quasi-drug composition or food composition containing the protein according to the present invention as an active ingredient can be taken before meals in addition to conventional ingestion.
  • the protein according to the present invention has the highest lactase activity at around 70 ° C. under the reaction conditions of pH 4.5 for 15 minutes (see Example 5 described later).
  • the protein according to the present invention has a molecular mass of about 140 kDa as determined by gel filtration, and a submass of about 86 kDa and about 50 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated into units (see Example 7 described below).
  • Km value (Michaelis constant) of the protein of the present invention is about 0.19 mM with respect to 2-nitrophenyl- ⁇ -D-galactopyranoside, 4-nitrophenyl- ⁇ -D-galacto
  • the Km value for pyranoside is about 1.2 mM
  • the Km value for lactose (measured using Glucose oxidase) is about 170 mM (see Example 8 described later).
  • a specific method for calculating the Km value is not particularly limited, and a known method can be freely selected and calculated. In the present invention, in particular, the Km value was calculated by the method described in Example 8 described later.
  • the protein according to the present invention does not decrease lactase activity for 3 hours or more at 37 ° C. at pH 4.5 and 7.0 (see Example 6 described later). That is, high lactase activity is maintained while digesting food and drink.
  • the protein according to the present invention does not show inactivation at pH 1.5 at 37 ° C. for 90 minutes, and remains about 70% active even after heating for 3 hours (see Example 6 described later). That is, high lactase activity is retained even when an enzyme preparation, pharmaceutical composition, quasi-drug composition or food composition containing the protein according to the present invention as an active ingredient is taken or ingested before a meal. .
  • the activity when heating for 60 minutes, at pH 2.0, the activity does not decrease up to 40 ° C., and 90% or more of the activity remains even at 50 ° C. (see Example 6 described later). Moreover, in pH 5.0, activity does not fall to 60 degreeC (refer Example 6 mentioned later). Furthermore, in the case of pH 7.0, the activity does not decrease up to 40 ° C., and about 60% of the activity remains even at 50 ° C. (see Example 6 described later).
  • the protein according to the present invention described above has characteristics unprecedented in the above-described physicochemical properties, so the origin is not particularly limited as long as it is a protein specified by the above-described physicochemical properties.
  • Examples of the present invention include those derived from microorganisms belonging to the genus Teratosphaeria. In this case, examples of microorganisms belonging to the genus Teratosphaeria include Teratosphaeria acidotherma.
  • Teratosphaeria acidotherma is a kind of fungus belonging to the Capnodiales family of Ascomycota (Ascomycota), and many exist as acidophilic bacteria in the soil of hot springs.
  • Ascomycota Capnodiales family of Ascomycota
  • Teratosphaeria acidotherma was isolated and purified from soil at a high temperature (approximately 40 ° C.) in acidic hot springs in Akita Prefecture and Iwate Prefecture. A specific screening method will be described in Example 1 described later.
  • amino acid structure is not limited as long as it is a protein specified by the above-mentioned physicochemical properties.
  • it can be specified by the following amino acid sequence.
  • the protein according to the present invention can be identified by the amino acid sequence represented by SEQ ID NO: 17.
  • the modified protein when a part of the amino acid sequence of a certain protein is modified, 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 protein function may be maintained before and after the modification. Therefore, the present invention provides, as another aspect, a protein having an amino acid sequence in which one to several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 17, and having lactase activity. . “Deletion, substitution and / or addition of one to several amino acids constituting an amino acid sequence” typically refers to a difference in a part of the amino acid sequence.
  • 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 the entire amino acid sequence, preferably a number corresponding to less than about 20%, and more preferably a number corresponding to less than about 10%. Even more preferably a number corresponding to less than about 5%, most preferably a number corresponding to less than about 1%. That is, 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 identity with the amino acid sequence of SEQ ID NO: 17. It means having.
  • a method of obtaining a protein by causing a conservative amino acid substitution at an amino acid residue that is not essential for lactase activity is preferred.
  • 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.
  • the amino acid residue is composed of a basic side chain (for example, lysine, arginine, histidine), an acidic side chain (for example, aspartic acid, glutamic acid), an uncharged polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, Tyrosine, cysteine), nonpolar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chains (eg threonine, valine, isoleucine), aromatic side chains (eg tyrosine, phenylalanine) , Tryptophan, and histidine).
  • a conservative amino acid substitution is preferably a substitution between amino acid residues within the same family.
  • the protein according to the present invention may be a part of a larger protein (for example, a fusion protein).
  • a fusion protein for example, 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.
  • Gene, Recombinant Vector, Transformant> (1) Gene in the present invention, a gene encoding the protein is provided.
  • the gene of the present invention comprises DNA encoding the amino acid sequence of SEQ ID NO: 17.
  • a specific example of this aspect is DNA consisting of the nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 20.
  • 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.
  • a DNA encoding a protein having a base sequence equivalent to the base sequence of SEQ ID NO: 16 or 20 and having lactase activity hereinafter also referred to as “equivalent DNA”.
  • the “equivalent base sequence” here is partly different from the nucleic acid shown in SEQ ID NO: 16 or SEQ ID NO: 20, but the function of the protein encoded by the difference (in the present invention, lactase activity) is substantially reduced. This means a base sequence that is not affected by any significant influence.
  • a specific example of equivalent DNA is DNA that hybridizes under stringent conditions to a base sequence complementary to the base sequence of SEQ ID NO: 16 or SEQ ID NO: 20.
  • 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, for example, Molecular Cloning (Third Edition, Cold Spring Harbor Press, New York, Current Protocols in Molecular Biological. Can be set with reference to.
  • a hybridization solution (50% formamide, 10 ⁇ SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0), 5 ⁇ Denhardt solution, 1% SDS, 10% dextran sulfate, 10 ⁇ g / Incubate at about 42 ° C. to about 50 ° C. with ml of denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5), and then at about 65 ° C. with 0.1 ⁇ SSC, 0.1% SDS.
  • the conditions for washing at about 70 ° C. can be mentioned.
  • More 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 as a hybridization solution.
  • the conditions using 10 ⁇ g / ml denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)) can be mentioned.
  • equivalent DNA include substitution, deletion, insertion, addition, or inversion of one or more (preferably 1 to several) bases based on the base sequence shown in SEQ ID NO: 16 or SEQ ID NO: 20.
  • Examples thereof include DNA encoding a protein having a base sequence and having lactase activity. 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, DNA ligase, etc., position-directed mutagenesis (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Press, New York, suddenly) Including base substitution, deletion, insertion, addition, and / or inversion using mutation introduction methods (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Press, New York), etc. using mutation introduction methods (Molecular Cloning, Third Edition, Chapter 13, Cold Spring Harbor Press, New York).
  • DNA having the nucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 20 is modified. It can be obtained by Rukoto.
  • the equivalent DNA can also be obtained by other methods such as ultraviolet irradiation.
  • DNA in which a difference in base 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 / primer capable of specifically hybridizing to the gene of the present invention from an appropriate Teratosphaeria ⁇ ⁇ ⁇ ⁇ ⁇ acidotherma genomic DNA library or cDNA library, or from an intracellular extract of Teratosphaeria acidotherma. It can be prepared using as appropriate. Oligonucleotide probes and primers can be easily synthesized using a commercially available automated DNA synthesizer. For the method of preparing the 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 nucleotide sequence of SEQ ID NO: 16 or SEQ ID NO: 20 can be isolated using a hybridization method using the whole or a part of the nucleotide 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: 17 and the information on the base sequence of SEQ ID NO: 16 or SEQ ID NO: 20, a target gene can also be obtained by chemical synthesis (Reference: Gene, 60 (1), 115-127 (1987)).
  • a protein according to the present invention (protein having lactase activity) is isolated and purified from Teratosphaeria® acidotherma, and information on the partial amino acid sequence is obtained.
  • an amino acid sequence analysis [protein-sequencer 476A] is performed by the Edman degradation method [Journal of Biological Chemistry, Vol. 256, pp. 7990-7997 (1981)] according to a conventional method. , 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 gene encoding the protein according to the present invention is cloned.
  • cloning can be performed using a hybridization method or PCR.
  • the hybridization method for example, the method described in Molecular Cloning (Third Edition, Cold Spring HarborLaboratory Press, New York) can be used.
  • a genomic DNA of a microorganism that produces the protein according to the present invention is used as a template, and a PCR reaction is performed using a synthetic oligonucleotide primer designed based on partial amino acid sequence information to obtain a target gene fragment.
  • the PCR method can be performed, for example, according to the method described in PCR technology [PCR Technology, Erlich HA editing, 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 partial amino acid sequence of the protein according to the present invention is included. The corresponding sequence is found and a part of the gene of interest can be obtained.
  • the gene encoding the protein according to the present invention can be cloned by further performing a hybridization method or the like using the obtained gene fragment as a probe.
  • the whole or part of the gene according to the present invention (SEQ ID NO: 16 or 20) whose entire base sequence has been clarified is used as a probe for hybridization, whereby a genomic DNA of a microorganism that produces a protein having other lactase activity. It is also possible to select DNA having high homology with the gene of SEQ ID NO: 16 or SEQ ID NO: 20 from the library or cDNA library.
  • PCR primers can be designed. By performing a PCR reaction using this primer, a gene fragment highly homologous to the gene of SEQ ID NO: 16 or SEQ ID NO: 20 can be detected, and further the entire gene can be obtained.
  • the gene according to the present invention can be used by inserting it into an appropriate vector.
  • the type of vector that can be used in the present invention refers to a nucleic acid molecule that can transport a nucleic acid molecule inserted therein into a target such as a cell, and the type and form thereof are not particularly limited.
  • 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 according to the purpose of use (cloning, protein expression) and in consideration of the type of host cell.
  • specific examples of the vector include vectors having E. coli as a host (M13 phage or a variant thereof, ⁇ phage or a variant thereof, pBR322 or a variant 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 Spring Harbor Laboratory Press, New York, etc.).
  • Transformant A transformant can be prepared by introducing the recombinant vector according to the present invention into an appropriate host.
  • 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 (Felner) , P.L. et al., Proc.Natl.Acad.Sci.U.S.A.
  • the host cell that can be used in the present invention is not particularly limited as long as the protein having lactase activity of the present invention is expressed.
  • yeasts it is more preferable to select yeasts belonging to the genus Saccharomyces, Schizosaccharomyces, and Pichia.
  • An example is Pichia pastoris.
  • filamentous fungi it is more preferable to select filamentous fungi belonging to the genus Aspergillus, and examples of the genus Aspergillus include Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus awamori.
  • Enzyme preparation, pharmaceutical composition, quasi-drug composition> The protein according to the present invention can be suitably used for enzyme preparations, pharmaceutical compositions and quasi-drug compositions by utilizing its excellent lactase activity.
  • the specific dosage form is not particularly limited as long as it is a dosage form taken internally or orally, and can be applied to any dosage form. .
  • it can be applied to oral preparations such as powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts and pills.
  • one or more pharmacologically acceptable additives can be freely selected and contained.
  • excipients binders, disintegrants, surfactants, preservatives, colorants, flavoring agents, fragrances, stabilizers, preservatives, antioxidants, etc.
  • Additives can be included.
  • a sustained-release preparation can be obtained using a drug delivery system (DDS).
  • DDS drug delivery system
  • the enzyme preparation, the pharmaceutical composition and the quasi-drug composition according to the present invention can be used as a mixture by freely selecting any one or more existing drugs.
  • drugs such as antacids, gastric agents, digestive agents, intestinal regulating agents, H2 blockers, proton pump inhibitors, antipruritic agents, analgesic / spasmodic agents, laxatives, laxatives, and the like can be added.
  • the protein which concerns on this invention can be used suitably for a food composition using the outstanding lactase activity.
  • milk, juice, sports drinks, beverages such as tea, coffee, tea, seasonings such as soy sauce, soups, creams, various dairy products, ice cream and other frozen desserts, various powdered foods (including beverages),
  • beverages such as tea, coffee, tea, seasonings such as soy sauce, soups, creams, various dairy products, ice cream and other frozen desserts, various powdered foods (including beverages)
  • It can be used for all foods and beverages such as processed foods such as foods for preservation, frozen foods, breads and confectionery.
  • health functional foods including specific health functional foods, nutritional functional foods and beverages
  • so-called health foods including beverages
  • concentrated nutrients liquid foods, infant and infant foods.
  • livestock mammals such as cattle, horses and pigs, poultry such as chickens and quails, pets such as reptiles, birds and small mammals, and farmed fish.
  • the food composition according to the present invention in addition to the protein according to the present invention, it is possible to freely select one or two or more components that can be usually used in foods and drinks and mix them.
  • various seasonings, excipients, buffers, suspending agents, preservatives, emulsifiers, stabilizers, fragrances, fats and oils, brighteners, thickeners, colorants, preservatives, binders, binder reinforcing agents , Emulsion additives, pH adjusters, and the like which can be used in the food field.
  • excipient for example, starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like can be used.
  • buffer for example, phosphate, citrate, acetate and the like can be used.
  • stabilizer for example, propylene glycol, ascorbic acid or the like can be used.
  • Examples of the pH adjuster include organic acids such as itaconic acid, succinic acid, tartaric acid, fumaric acid, citric acid, malic acid, adipic acid, gluconic acid, pyrophosphoric acid, acetic acid, lactic acid, ⁇ -ketoglutaric acid, phytic acid, and the like.
  • Organic acid salts; inorganic acids such as carbonic acid or inorganic acid salts; acidic amino acids such as aspartic acid and glutamic acid; basic amino acids such as arginine, lysine and histidine can be used.
  • fragrance examples include animal fragrances such as musk, civet, castorium and ambergris; anise essential oil, angelica essential oil, ylang ylang essential oil, iris essential oil, fennel essential oil, orange essential oil, caranga essential oil, caraway essential oil, cardamom essential oil, guayakwood essential oil , Cumin essential oil, black letter essential oil, cinnamon essential oil, cinnamon essential oil, geranium essential oil, copaiba balsam essential oil, coriandel essential oil, perilla essential oil, cedarwood essential oil, citronella essential oil, jasmine essential oil, gingergrass essential oil, cedar essential oil, spearmint essential oil, western peppermint essential oil , Otsuka perfume essential oil, tuberose essential oil, clove essential oil, orange flower essential oil, winter green essential oil, trout balsam essential oil, buttery essential oil, rose essential oil, palmarosa essential oil, persimmon essential oil, hiba essential oil, sandalwood essential oil, petitgren essential oil, bay
  • natural polymers or starch-based or cellulose-based natural polymer derivatives can be used.
  • natural polymer include seaweed extracts such as fucoidan and carrageenan, seed exudates such as guar gum, resin-like adhesives such as gum arabic, and microorganism-generated adhesive substances such as xanthan gum.
  • starch-based or cellulose-based natural polymer derivative include starch-based starch-based starch-based or cellulose-based natural polymer derivatives such as methylcellulose.
  • oils and fats include avocado oil, linseed oil, almond oil, fennel oil, sesame oil, olive oil, orange oil, orange rafa oil, cacao oil, chamomile oil, carrot oil, cucumber oil, coconut oil, sesame oil, rice oil, Safflower oil, shea fat, liquid shea fat, soybean oil, camellia oil, corn oil, rapeseed oil, persic oil, castor oil, sunflower oil, camellia seed oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, Palm oil, palm kernel oil, owl, coconut oil, beef tallow, lard, etc. can be used.
  • oils and fats modified by hydrogenation, fractionation, transesterification and the like can be used.
  • brighteners include waxes such as beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax, squalene, squalane, pristane and the like (vegetable, animal It does not matter)
  • Mineral oils such as liquid paraffin, petrolatum, paraffin, ozokelide, ceresin, and microcristan wax can be used.
  • the binder for example, soy protein, egg protein, milk protein, casein, starch, transglutaminase and the like can be used.
  • additives include, for example, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil Natural fatty acids such as lanolin fatty acid; fatty acids such as isononanoic acid, caproic acid, 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, isopentanoic acid and the like.
  • the production method according to the present invention is a method for collecting a protein having lactase activity from a culture obtained by culturing a microorganism capable of producing the protein according to the present invention or a transformant according to the present invention in a nutrient medium. is there.
  • the microorganism that can be used in the production method according to the present invention is not particularly limited as long as it is a microorganism having the above-described physicochemical properties and the ability to produce a protein having lactase activity, and a known microorganism can be freely selected and used. be able to.
  • a microorganism belonging to the genus Teratosphaeria can be mentioned.
  • examples of microorganisms belonging to the genus Teratosphaeria include Teratosphaeria acidotherma.
  • the microorganism used in the production method according to the present invention is not limited to wild strains, even if the wild strain is a mutant strain mutated by artificial mutation means using ultraviolet rays, X-rays, radiation, various drugs, etc. It can be used as long as it has the ability to produce an acid-resistant enzyme having the aforementioned lactase activity.
  • lactose is preferably used as an inducer.
  • the carbon source of the culture medium which can be used is not specifically limited,
  • the carbon source used for a well-known culture medium can be freely selected 1 type or 2 types or more and can be used.
  • the nitrogen source is not particularly limited, and one or more nitrogen sources used in a known medium can be freely selected and used.
  • inorganic nitrogen sources such as ammonium sulfate, ammonium nitrate, phosphate-ammonium phosphate, diammonium phosphate, ammonium chloride, corn gluten meal, soy flour, casamino acid, coffee lees, cottonseed oil lees, yeast extract, malt extract, corn steep liquor
  • organic nitrogen raw materials such as casein hydrolyzate, bran, meat extract, amino acid, and peptone can be used.
  • the inorganic nutrient source is not particularly limited, and one or more inorganic nutrient sources used in known media can be freely selected and used.
  • one or more inorganic nutrient sources used in known media can be freely selected and used.
  • in addition to sodium, magnesium, potassium, iron, zinc, calcium, manganese salts, vitamins and the like can be mentioned.
  • the specific temperature for culturing in the production method according to the present invention is not particularly limited, and can be freely set as long as the effects of the present invention are not impaired. In the present invention, it is particularly preferably carried out in a temperature range of 25 to 45 ° C., more preferably in a temperature range of 28 to 32 ° C., and most preferably at 30 ° C.
  • the pH of the medium is not particularly limited and can be freely set as long as the effects of the present invention are not impaired.
  • the pH is preferably set to 3.0 to 5.0, more preferably set to pH 3.8 to 4.2, and most preferably set to pH 4.0.
  • the culture period in the production method according to the present invention is not particularly limited, and can be freely set according to the bacterial cell concentration, the medium pH, the medium temperature, the structure of the medium and the like as long as the effects of the present invention are not impaired.
  • the culture period is preferably set to 2 to 5 days, more preferably 3 to 4 days.
  • the culture method for example, stationary culture, shaking culture, and aerobic deep culture using jar fermenter can be used.
  • the protein of the present invention is purified and recovered.
  • the protein purification / recovery method is not particularly limited, and any known method can be freely selected. For example, after culturing the microbial cells, the microbial cells are collected by filtration, etc., crushed, etc. to prepare a crude enzyme solution, concentrated and desalted, and then subjected to various chromatography (ion exchange column chromatography). Chromatography, brine column chromatography, affinity column chromatography, gel filtration column chromatography, etc.) can be performed sequentially to obtain the protein of the present invention.
  • the transformant is cultured under conditions where a protein encoded by the gene introduced into the transformant is produced.
  • 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 is recovered. About collection
  • the lactase activity was measured by the following method. After 2 mL of 12.3 mM 2-nitrophenyl- ⁇ -D-galactopyranoside (ONPG) solution was allowed to stand at 37 ° C. for 10 minutes, 0.5 mL of the sample solution was added and immediately mixed. This solution was allowed to stand at 37 ° C. for exactly 15 minutes, and then 2.5 mL of 10 g / dL sodium carbonate solution was added and mixed to stop the reaction. Further, 20 mL of water was added and mixed. For the prepared mixed solution, the absorbance at an absorption wavelength of 420 nm was measured using water as a control.
  • ONPG 2-nitrophenyl- ⁇ -D-galactopyranoside
  • A1 Absorbance of sample A2: Absorbance of blank n: Dilution factor
  • Example 1 Cell separation> Diluted suspension of soil at high temperature (approximately 40 ° C) in acid hot springs in Akita and Iwate Prefectures was applied to glucose-containing agar medium at pH 1 or pH 2.5 and cultured at 42-45 ° C for 10 days. . Thereafter, the grown strain was isolated.
  • the isolated strain was cultured at 30 ° C. using a lactose-containing medium having a pH of 4.5.
  • the cultured cells were crushed to prepare a crude enzyme solution.
  • the lactase activity of the crude enzyme solution was measured at pH 1.5, pH 4.5, and pH 7.0, and then the activity ratio at each pH was determined.
  • the crude enzyme solution was heated at 37 ° C. for 3 hours at pH 1.5 and pH 5.0 to examine acid resistance.
  • the strain selected as an excellent strain as described above was subjected to liquid culture, disrupted with bacterial cells, and a crude enzyme solution was prepared.
  • This crude enzyme solution was partially purified by DEAE-Toyopearl (registered trademark, manufactured by Tosoh Corporation) column chromatography.
  • the lactase activity of each fraction of column chromatography was measured at pH 1.5, pH 4.5 and pH 7.0, and the pH activity ratios were summarized.
  • the acid resistance was examined by heating at 37 ° C. for 3 hours at pH 1.5 and pH 5.0.
  • the rRNA of the strain obtained by screening was extracted and the strain was identified.
  • the strain obtained by screening was identified as Teratosphaeria acidotherma.
  • the gene sequence of the D2 region of the obtained strain is shown in SEQ ID NO: 1
  • the gene sequence of the ITS region is shown in SEQ ID NO: 2
  • the gene sequence of the D2 region of Teratosphaeria acidotherma is shown in SEQ ID NO: 3
  • the gene sequence of the ITS region is shown in SEQ ID NO: 4, respectively. .
  • Example 2 Purification of acid-resistant protein having lactase activity> (1) Culture First, in order to culture Teratosphaeria acidotherma obtained in Example 1, liquid media shown in Table 1 below were prepared.
  • the 500 mL Sakaguchi flask liquid medium prepared as described above was inoculated with the Teratosphaeria acidotherma spore suspension obtained in Example 1 and precultured. After culturing at 30 ° C. and a rotation speed of 120 rpm for 2 days, 100 mL of the preculture solution was inoculated into a new 3 L flat bottom flask liquid medium to perform main culture. After culturing at 30 ° C. and a rotation speed of 115 rpm for 4 days, the cells were collected by filtration with filter paper.
  • Example 3 Examination of optimum pH> The protein solution purified in Example 2 and the following pH buffer solutions were mixed in equal amounts, reacted at 37 ° C. for 15 minutes, and lactase activity was measured. pH 1.0 to pH 4.5: 0.1 M sodium acetate-hydrochloric acid buffer pH 4.0 to pH 5.5: 0.1 M sodium acetate-acetic acid buffer pH 5.5 to pH 8.0: 0.1 M potassium phosphate buffer
  • the protein according to the present invention was found to have the highest lactase activity around pH 3.0 to 4.0. It was also confirmed that the lactase activity was at least 80% at the optimum pH at pH 4 to 5, and at least 40% at the optimum pH at pH 1.0 and pH 7.0.
  • Example 4 Examination of stable pH range> The protein solution purified in Example 2 and each pH buffer solution used in Example 3 were mixed in equal amounts and incubated at 50 ° C. for 60 minutes, and then the remaining lactase activity was measured at pH 4.5.
  • the protein according to the present invention was confirmed to be stable at pH 1.5 to 7.0.
  • Example 5 Examination of optimum temperature> Equal amounts of the protein solution purified in Example 2 and 0.1M sodium acetate-acetate buffer at pH 4.5 were mixed and reacted at 30-80 ° C. and pH 4.5 for 15 minutes, and lactase activity was measured.
  • the protein according to the present invention was found to have the highest lactase activity around 70 ° C.
  • Example 6 Examination of stable temperature range> The protein solution purified in Example 2 above, a pH 1.5 0.1M sodium acetate-hydrochloric acid buffer solution, a pH 4.5 0.1M sodium acetate-acetic acid buffer solution, and a pH 7.0 0.1M potassium phosphate buffer solution. After mixing in equal amounts and incubating at 37 ° C. for 0-3 hours, the remaining lactase activity was measured at pH 4.5. Similarly, the protein solution purified in Example 2 above, 0.1M sodium acetate-hydrochloric acid buffer solution at pH 2.0, 0.1M sodium acetate-acetic acid buffer solution at pH 5.0, 0.1M phosphoric acid solution at pH 7.0. The remaining lactase activity after mixing each with an equal amount of potassium buffer and heating at 0 to 60 ° C. for 60 minutes was also examined.
  • Results are shown in FIG. 4 and FIG. As shown in FIG. 4, it was found that the protein according to the present invention did not decrease lactase activity for 3 hours or more at 37 ° C. at pH 4.5 and pH 7.0. That is, it was found that high lactase activity was maintained during digestion of food and drink.
  • the protein according to the present invention was not inactivated for 90 minutes at 37 ° C. at pH 1.5, and about 70% of the activity remained even after heating for 3 hours. That is, high lactase activity is retained even when an enzyme preparation, pharmaceutical composition, quasi-drug composition or food composition containing the protein according to the present invention as an active ingredient is taken or ingested before a meal. I understood that.
  • Example 7 Measurement of molecular mass> The protein purified in Example 2 was calculated to have a molecular mass of about 140 kDa by gel filtration. In addition, when the protein purified in Example 2 was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two bands indicating the protein appeared (FIG. 6), and the protein according to the present invention was found to consist of two subunits. Furthermore, from the results of SDS-PAGE, it was confirmed that the molecular masses of the two subunits were about 86 kDa and about 50 kDa.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • Example 8 Measurement of Km value>
  • the concentrations of 2-nitrophenyl- ⁇ -D-galactopyranoside (ONPG), 4-nitrophenyl- ⁇ -D-galactopyranoside (PNPG), and lactose were changed. Lactase activity was measured, and each Michaelis constant (Km) was determined from the Hanes-Woolf plot.
  • Km Michaelis constant
  • the Km value for 2-nitrophenyl- ⁇ -D-galactopyranoside was 0.19 mM
  • the Km value for 4-nitrophenyl- ⁇ -D-galactopyranoside was 1.2 mM
  • lactose Glucose oxidase was used. The Km value for the measurement was found to be 170 mM.
  • Example 9 Examination of effects of various chemical substances and metals on enzymes>
  • various chemical substances and metals shown in Table 2 below were added to the reaction solution so as to be 1 mM, and reacted at 37 ° C. for 15 minutes at pH 4.5 to measure lactase activity. .
  • the results are shown in Table 2.
  • Example 10 Examination of substrate specificity> Regarding the substrate specificity of the protein purified in Example 2, 2-nitrophenyl-bD-galactopyranoside (ONPG), 4-nitrophenyl-bD-galactopyranoside, 4-nitrophenyl-bD-fucopyranoside, 4-nitrophenyl-bD-glucopyranoside, 4 -Nitrophenyl-bD-xylopyranoside, 4-nitrophenyl-bD-mannopyranoside, 4-nitrophenyl-bD-galactopyranoside, 4-nitrophenyl-bD-glucopyranoside, 4-nitrophenyl-bL-arabinofuranoside, and 4-nitrophenyl-bD-mannopyranoside were studied. The results are shown in Table 3.
  • Example 11 Determination of Partial Amino Acid Sequence>
  • the protein purified in Example 2 was subjected to SDS-PAGE, and the internal amino acid sequence (SEQ ID NO: 5) of the band of about 86 kDa and the N-terminal sequence (SEQ ID NO: 6) of about 50 kDa were analyzed.
  • Example 2 The protein purified in Example 2 was subjected to SDS-PAGE and then blotted onto a PVDF membrane. A protein band of about 86 kDa confirmed by CBB staining was treated with trypsin. Protein digests were separated by HPLC, and the amino acid sequence of one part of the obtained protein peak was analyzed (SEQ ID NO: 5). Further, the N-terminal amino acid sequence of the approximately 50 kDa protein band confirmed by CBB staining was analyzed (SEQ ID NO: 6).
  • Example 12 Acquisition of cDNA Fragment> Using the amino acid sequence information obtained in Example 11, cDNA encoding the protein purified in Example 2 was obtained.
  • SEQ ID NO: 5 Based on the internal amino acid sequence (SEQ ID NO: 5) of the band of about 86 kDa, a Degenerate Primer (Primer (1)) of SEQ ID NO: 7 was prepared.
  • the template was prepared from Teratosphaeria acidotherma.
  • the cells of Teratosphaeria acidotherma obtained by liquid culture were collected and frozen, and then dried by lyophilization. Beads were added to the dried cells, and the cells were crushed with a bead shocker. Genomes were collected from the crushed cells using DNeasy Plant Mini Mini Kit (manufactured by QIAGEN). This Genome was used as a template.
  • the PCR reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplified DNA band was confirmed under UV.
  • a DNA band of about 1,600 bp was cut out and DNA was extracted from an agarose gel.
  • the extracted DNA was TA cloned using pGEM-T-Easy Vector Systems (manufactured by Promega).
  • E. coli JM109 was transformed with the TA cloning reaction solution to obtain a plasmid introduced with the target DNA.
  • the base sequence of the DNA introduced into the plasmid was decoded, and the partial base sequence of the gene encoding the protein purified in Example 2 was revealed.
  • Teratosphaeria acidotherma was cultured as follows using the medium shown in Table 1 above.
  • the prepared 500 mL Sakaguchi flask liquid medium was inoculated with Teratosphaeria acidotherma spore suspension and precultured. After culturing at 30 ° C. and a rotation speed of 120 rpm for 2 days, 100 mL of the preculture solution was inoculated into a new 3 L flat bottom flask liquid medium to perform main culture. After culturing at 30 ° C. and a rotation speed of 115 rpm for 1 day, the cells were collected by filtration with filter paper.
  • reaction compositions shown in Table 6 below were prepared, and reacted at 65 ° C. for 5 minutes. After the reaction, it was cooled to 4 ° C.
  • PCR was performed using Primer (3) and Primer (4) under the composition shown in Table 9 below and the reaction conditions shown in Table 10 below. Amplified.
  • DNA-Polymerase TaKaRa-Taq manufactured by Takara Bio was used.
  • the PCR reaction solution was subjected to agarose electrophoresis, stained with ethidium bromide, and the amplified DNA band was confirmed under UV.
  • a DNA band of about 2,000 bp was cut out and DNA was extracted from an agarose gel.
  • the extracted DNA was TA cloned using pGEM-T-Easy Vector Systems (manufactured by Promega).
  • E. coli JM109 was transformed with the TA cloning reaction solution to obtain a plasmid introduced with the target DNA. Decoding the base sequence of the DNA introduced into the plasmid, revealing that the introduced DNA is a partial fragment of the cDNA containing the 3 ′ end region of the gene encoding the protein purified in Example 2 above. did.
  • 5′-RACE method was used to determine the 5 ′ terminal nucleotide sequence further upstream from the obtained gene partial sequence.
  • 5′-Full RACE Core Set manufactured by Takara Bio Inc.
  • reaction was carried out under the reaction composition A shown in Table 11 below and the reaction conditions shown in Table 12 below.
  • Reaction composition B shown in Table 13 below was prepared using the reaction composition A after the reaction. About this reaction composition B, reaction was performed at 30 degreeC for 1 hour.
  • Reaction composition C shown in Table 14 below was added to the DNA recovered by ethanol precipitation, and the reaction was carried out at 15 ° C. for 15 hours.
  • PCR was carried out under the reaction conditions shown in Table 15 below and the reaction conditions shown in Table 16 below using Primer® (6) and Primer® (7) with the reaction composition C after the completion of the reaction as a template.
  • Primer® (6) and Primer® (7) for DNA-Polymerase, TaKaRa-LA-Taq manufactured by Takara Bio was used.
  • PCR was further performed under the reaction conditions shown in Table 17 below and the reaction conditions shown in Table 18 below, using Primer D (8) and Primer (9) using the reaction composition D after the reaction as a template.
  • Primer D (8) and Primer (9) using the reaction composition D after the reaction as a template.
  • primer D (8) and Primer (9) using the reaction composition D after the reaction as a template.
  • TaKaRa-LA-Taq manufactured by Takara Bio was used.
  • the PCR reaction solution with the above reaction composition E was subjected to agarose electrophoresis, stained with ethidium bromide, and the DNA band amplified under UV was confirmed. An approximately 1,800 bp DNA band was cut out and DNA was extracted from an agarose gel. The extracted DNA was TA cloned using pGEM-T-Easy Vector Systems (Promega). E. coli JM109 was transformed with the TA cloning reaction solution to obtain a plasmid introduced with the target DNA. The base sequence of the DNA introduced into the plasmid is decoded, and it is revealed that the introduced DNA is a partial fragment of cDNA containing the 5 ′ end region of the gene encoding the protein purified in Example 2 above. did.
  • the base sequences of the gene encoding the protein obtained above were aligned, and the full-length base sequence (SEQ ID NO: 16) of the gene encoding the protein and the amino acid sequence (SEQ ID NO: 17) of the protein were determined.
  • ⁇ Experimental Example 14 Determination of nucleotide sequence in Genome> Based on the information of the full-length base sequence (SEQ ID NO: 16) obtained in Experimental Example 13, Primer (10) of SEQ ID NO: 18 and Primer (11) of SEQ ID NO: 19 were designed. Using the Teratosphaeria acidotherma Genemo as a template, PCR was performed using Primer (10) (SEQ ID NO: 18) and Primer (11) (SEQ ID NO: 19) to determine the nucleotide sequence of the gene encoding the protein in the genome. (SEQ ID NO: 20).
  • the protein according to the present invention is stable in the stomach on an empty stomach and exhibits high activity in the stomach after a meal. Therefore, it is possible to take an enzyme preparation, pharmaceutical composition, quasi-drug composition or food composition containing this as an active ingredient before meals, and also has a very high lactose-degrading action even after meals. Can be realized. As a result, the range of options for taking an enzyme preparation or the like in lactose intolerance is widened, and symptoms can be effectively suppressed by lactose intolerance.

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Abstract

L'invention concerne une nouvelle protéine ayant une activité lactase, qui est stable à une valeur de pH allant de 1 à 2 et est hautement active à une valeur de pH allant de 4 à 5. La présente invention concerne une protéine ayant une activité lactase, qui est la plus active à une valeur de pH allant d'environ 3,0 à 4,0 et est stable dans une plage de pH allant de 1,5 à 7,0. La protéine selon la présente invention est stable dans l'estomac au cours de l'état à jeun et devient hautement active dans l'estomac pendant et après l'ingestion de repas. Par conséquent, une préparation enzymatique, une composition de médicament, une composition de quasi-médicament ou une composition alimentaire comprenant la protéine en tant que principe actif peut être ingérée avant les repas et peut présenter une activité de décomposition du lactose extrêmement élevée pendant et après l'ingestion de repas.
PCT/JP2013/050559 2012-05-10 2013-01-15 Protéine ayant une activité lactase, gène codant pour ladite protéine, vecteur recombinant portant ledit gène, transformant, procédé de fabrication de ladite protéine, et utilisation de ladite protéine WO2013168438A1 (fr)

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WO2023106420A1 (fr) * 2021-12-09 2023-06-15 合同酒精株式会社 Solution de lactase
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WO2016088589A1 (fr) * 2014-12-05 2016-06-09 合同酒精株式会社 Solution de lactase et produit laitier l'utilisant
CN107109384A (zh) * 2014-12-05 2017-08-29 合同酒精株式会社 乳糖酶溶液及使用其的乳制品
JPWO2016088589A1 (ja) * 2014-12-05 2017-11-30 合同酒精株式会社 ラクターゼ溶液及びそれを用いた乳製品
US10368558B2 (en) 2014-12-05 2019-08-06 Godo Shusei Co., Ltd. Lactase solution and dairy product using same
CN107109384B (zh) * 2014-12-05 2021-06-08 合同酒精株式会社 乳糖酶溶液及使用其的乳制品
US11905232B2 (en) 2019-02-08 2024-02-20 Nogra Pharma Limited Process of making 3-(4′-aminophenyl)-2-methoxypropionic acid, and analogs and intermediates thereof
WO2023106420A1 (fr) * 2021-12-09 2023-06-15 合同酒精株式会社 Solution de lactase
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