Allulose amount increasing agent

Abstract

Provided is a technique for increasing the amount of allulose in a raw material or a fructose-containing food or beverage.　The present technology provides an allulose amount increasing agent comprising a D-allulose-3-epimerase. The allulose amount increasing agent may further contain a glucosyltransferase. The present technology also provides a method for producing an allulose-containing food or beverage and a method for increasing the amount of allulose in a raw material or a fructose-containing food or beverage, each of the methods comprising a step for allowing a D-allulose-3-epimerase to act on a raw material or a fructose-containing food or beverage.

Description

Allulose bulking agent

[0001] The present technology relates to an allulose extender, more specifically to an allulose extender using a specific enzyme, an allulose-containing food or drink using the allulose extender, a method for producing an allulose-containing food or drink, and a method for increasing allulose in a fructose-containing food or drink.

[0002] In recent years, in response to the health-conscious social trend, the food market has seen the development of products that claim to be low in calories. For example, fruit juice drinks, vegetable juice drinks, and mixed juice drinks are widely used as sources of fruit and vegetable intake due to their convenience, and are preferred by many consumers for the purpose of maintaining health. However, these drinks contain carbohydrates derived from fruits and vegetables, and from the perspective of reducing calories, it is desirable to reduce the carbohydrate content as much as possible.

[0003] For example, Patent Document 1 discloses a method for producing low-calorie juice, which involves treating fruit juice with an ultrafiltration membrane to retain cloudy fruit juice, which has concentrated sugars, and allowing clear fruit juice with reduced sugar content to pass through the membrane. Patent Document 2 also discloses a method for producing vegetable or fruit juice, which involves adding lactic acid bacteria to vegetable or fruit juice, growing the lactic acid bacteria, and reducing the sugar concentration in the vegetable or fruit juice through lactic acid fermentation. However, membrane treatment of fruit juice, for example, has the problem of losing the richness and richness of the juice due to the removal of sugars. Furthermore, techniques for growing lactic acid bacteria and reducing calories through lactic acid fermentation require sterilization and alcohol removal processes, which necessitate a technology that facilitates subsequent processes.

[0004] Special table publication No. 2015-522294 Publication of Japanese Patent Application Publication No. 10-271980

[0005] As mentioned above, technologies for reducing the calorie content of foods and beverages are being developed, but these technologies are still in the development stage.

[0006] Therefore, the main objective of this technology is to provide a technology for increasing the amount of allulose in raw materials or fructose-containing food and beverages.

[0007] The present technology first provides an allulose extender containing D-allulose-3-epimerase. The D-allulose-3-epimerase may be derived from Asaia krungthepensis. The allulose extender of the present technology may further contain glucosyltransferase. The present technology also provides allulose-containing foods and beverages that use the allulose extender.

[0008] The present technology also provides a method for producing allulose-containing foods and beverages, which includes a step of treating a raw material or a fructose-containing food and beverage with D-allulose-3-epimerase. The method for producing allulose-containing foods and beverages according to the present technology may further include a step of treating the raw material or the fructose-containing food and beverage with glucosyltransferase.

[0009] Furthermore, the present technology also provides a method for increasing the allulose content of fructose-containing foods and beverages, which comprises the step of treating a raw material or a fructose-containing food and beverage with D-allulose-3-epimerase.The method for increasing the allulose content of fructose-containing foods and beverages according to the present technology may further comprise the step of treating the raw material or the fructose-containing food and beverage with glucosyltransferase.

[0010] In addition, the present technology also provides D-allulose-3-epimerase with excellent allulose-producing ability.

[0011] The technical terms used in this technology will be explained below.

[0012] <Amino Acid Notation> The 20 types of amino acid residues in an amino acid sequence may be represented by a single letter abbreviation: G for glycine (Gly), A for alanine (Ala), V for valine (Val), L for leucine (Leu), I for isoleucine (Ile), F for phenylalanine (Phe), Y for tyrosine (Tyr), W for tryptophan (Trp), S for serine (Ser), T for threonine (Thr), C for cysteine ​​(Cys), M for methionine (Met), D for aspartic acid (Asp), E for glutamic acid (Glu), N for asparagine (Asn), Q for glutamine (Gln), K for lysine (Lys), R for arginine (Arg), H for histidine (His), and P for proline (Pro).

[0013] <Amino Acid Sequence> In the present technology, the amino acid sequence displayed has the N-terminus at the left end and the C-terminus at the right end.

[0014] <Types of Amino Acids> In the present technology, "nonpolar amino acids" include alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, and tryptophan. "Uncharged amino acids" include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. "Acidic amino acids" include aspartic acid and glutamic acid. "Basic amino acids" include lysine, arginine, and histidine.

[0015] <Sequence Modifications (Substitution, Addition, Insertion, Deletion, Inversion, etc.)> In the present technology, modifications of amino acid sequences and / or nucleotide sequences include not only artificial modifications (substitution, addition, insertion, deletion, inversion, etc.), but also naturally occurring modifications (substitution, addition, insertion, deletion, inversion, etc.), i.e., cases where the sequences were originally different.

[0016] <Substitution> In this technology, "substitution" refers not only to the case where an amino acid residue substitution is artificially introduced, but also to the case where an amino acid residue substitution is naturally introduced, i.e., the amino acid residue is originally different. In this technology, the amino acid residue substitution may be artificial or natural, but artificial substitution is preferred. In addition, the amino acid residue at the mutation point (the amino acid residue to be substituted) is expressed as a combination of the above single letter representing the type of amino acid and a number representing the position of the amino acid. For example, if the 57th arginine is substituted with glycine, it is expressed as "R57G".

[0017] <Separation / Purification> In this technology, the term "separation" is used interchangeably with "purification." The term "separation" is used to distinguish it from the natural state, i.e., the state that exists in nature. The artificial operation of "separating" results in an "isolated state," which is a state that differs from the natural state. What is separated is clearly and decisively different from the natural product itself.

[0018] <Equivalent Sequence> In the present technology, an "equivalent sequence" refers to an amino acid sequence or a nucleotide sequence that differs in part from a reference amino acid sequence (in the present technology, SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 16, 17, 20, 21, 22, or 23) or nucleotide sequence (in the present technology, SEQ ID NOs: 2, 9, 10, 11, 12, 13, 14, 15, 18, or 19), but the difference does not substantially affect the function of the protein (in the present technology, D-allulose-3-epimerase activity or glucosyltransferase activity). Therefore, in the present technology, an enzyme having a polypeptide chain consisting of an equivalent amino acid sequence, or an enzyme encoded by a polynucleotide consisting of an equivalent nucleotide sequence, exhibits D-allulose-3-epimerase activity or glucosyltransferase activity.

[0019] Preferred embodiments for carrying out the present technology will be described below. Note that the embodiments described below are examples of typical embodiments of the present technology, and the scope of the present technology should not be construed as being narrow.

[0020] 1. Allulose extender The allulose extender according to the present technology contains D-allulose-3-epimerase, and may also contain glucosyltransferase, other enzymes, and other components as needed.

[0021] As shown in the Examples below, the present inventors have discovered that the amount of allulose in the allulose-containing food or drink produced can be increased by allowing D-allulose-3-epimerase to act on raw materials or fructose-containing food or drink during the production of allulose-containing food or drink.

[0022] Each component that can be used in the present technology will be described in detail below.

[0023] (1) D-Allulose-3-epimerase The D-allulose-3-epimerase that can be used in the present technology is an enzyme that catalyzes the epimerization of D-fructose as a substrate to produce allulose, and is a type of ketose-3-epimerase. The D-allulose-3-epimerase that can be used in the present technology may also be an enzyme that has other functions as long as it has D-allulose-3-epimerase activity. In the present technology, the use of D-allulose-3-epimerase to produce allulose-containing foods and beverages can exert the effect of increasing the amount of allulose.

[0024] The origin of D-allulose-3-epimerase that can be used in the present technology is not particularly limited, and examples include D-allulose-3-epimerase derived from microorganisms of the genus Asaia (e.g., Asaia krungthepensis, etc.), the genus Arthrobacter (e.g., Arthrobacter globiformis, etc.), and the genus Pseudomonas (e.g., Pseudomonas cichorii, etc.). Preferably, D-allulose-3-epimerase derived from a microorganism of the genus Asaia is used, and more preferably, D-allulose-3-epimerase derived from Asaia clungsapensis is used.

[0025] Allulose is classified as a ketose and is also called psicose. It is also called a rare sugar because it exists naturally but in small amounts. Allulose has a sweetness of about 70% of sucrose, but has 1 / 10 the calories of sucrose, and has been reported to have functions such as suppressing the rise in postprandial blood glucose levels and preventing obesity. Therefore, by increasing the amount of allulose in fructose-containing foods and beverages, a low-calorie effect can be achieved.

[0026] Here, "D-allulose-3-epimerase derived from Asaia clungsapensis" means a D-allulose-3-epimerase produced by a microorganism classified as Asaia clungsapensis (which may be a wild-type strain or a mutant strain), or a D-allulose-3-epimerase obtained by genetic engineering techniques using the D-allulose-3-epimerase gene. Therefore, a recombinant produced by a host microorganism into which the D-allulose-3-epimerase gene obtained from Asaia clungsapensis (or a modified version of said gene) has been introduced also falls under the category of "D-allulose-3-epimerase derived from Asaia clungsapensis."

[0027] The strain of Asaia krungthepensis is not particularly limited as long as it does not impair the action and effect of the present technology, and strains that are generally available from biological resource institutions and the like can be used.

[0028] In the present technology, microorganisms that produce enzymes that can be used in the present technology may be cultured, if necessary. Examples of microorganisms that produce D-allulose-3-epimerase include microorganisms of the genus Asaia, such as Asaia krungthepensis. Microorganisms that can be used may be wild-type strains or mutant strains (e.g., mutant strains induced by ultraviolet irradiation, etc.).

[0029] The culture conditions in the culture step can be appropriately set taking into consideration the properties of the microorganism to be cultured. A solid medium or a liquid medium can be used for culturing the microorganism. In addition, for industrial production, aeration and agitation culture can also be used.

[0030] As the nutrient source for the medium, substances required for the growth of microorganisms can be used as appropriate. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, molasses, or pyruvic acid. The nitrogen source may be any assimilable nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolysate, or alkaline extract of soybean meal. In addition to carbon and nitrogen sources, salts of phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, zinc, or the like; specific amino acids; specific vitamins, or the like, may also be used as needed.

[0031] The culture temperature may be appropriately set within a range in which the microorganism to be cultured can grow and the microorganism can produce the enzyme that can be used in the present technology. The culture temperature may be set, for example, to about 15°C to 37°C. The culture time may be, for example, about 12 hours to 48 hours. The culture step may be terminated, for example, when the enzyme that can be used in the present technology reaches its maximum yield.

[0032] The D-allulose-3-epimerase that can be used in the present technology can be prepared from the culture medium of the microorganism from which the D-allulose-3-epimerase is derived. Specific preparation methods include methods in which D-allulose-3-epimerase is recovered from the culture medium or cells of the microorganism. For example, when a D-allulose-3-epimerase-secreting microorganism is used, the cells can be recovered from the culture medium in advance by filtration, centrifugation, or the like, as necessary, and the enzyme can then be separated and / or purified. Furthermore, when a D-allulose-3-epimerase-non-secreting microorganism is used, the cells can be recovered from the culture medium in advance by pressure treatment, ultrasonic treatment, or the like, and the enzyme can then be extracted and / or separated and / or purified. Methods for enzyme separation and / or purification include known protein separation and / or purification methods, without particular limitation, and include, for example, centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins, etc. The isolated and / or purified enzyme can be powdered by a drying method such as freeze-drying or vacuum drying, or can be powdered using an appropriate excipient and / or drying aid in the drying method. The isolated and / or purified enzyme can also be liquefied by adding an appropriate additive and sterilizing by filtration.

[0033] In the present technology, specific examples of D-allulose-3-epimerase include the D-allulose-3-epimerase derived from Asaia clungsapensis shown below. For example, the amino acid sequence of D-allulose-3-epimerase derived from wild-type Asaia clungsapensis is shown in SEQ ID NO: 1. Furthermore, examples of DNA base sequences encoding the amino acid sequence shown in SEQ ID NO: 1 include the base sequences shown in SEQ ID NOs: 2 and 15. The base sequence shown in SEQ ID NO: 15 is the base sequence of DNA encoding wild-type D-allulose-3-epimerase derived from Asaia clungsapensis, and the base sequence shown in SEQ ID NO: 2 is the base sequence of SEQ ID NO: 15 modified to include codons suitable for Escherichia coli. Therefore, DNA in the present technology can be appropriately designed by those skilled in the art using the base sequence shown in SEQ ID NO: 2 as a reference sequence.

[0034] As other specific examples, the amino acid sequences of the modified D-allulose-3-epimerases disclosed in WO 2023 / 157936 are shown in SEQ ID NOS: 3 to 8. The base sequences of DNA encoding the amino acid sequences shown in SEQ ID NOS: 3 to 8 are shown in SEQ ID NOS: 9 to 14. The concept of "D-allulose-3-epimerase" as used herein also includes D-allulose-3-epimerases obtained by artificially substituting a portion of the amino acid sequence of the wild-type D-allulose-3-epimerase described above, as well as D-allulose-3-epimerases that have been naturally substituted to have such amino acid sequences. Therefore, the concept of "D-allulose-3-epimerase" as used herein also includes the modified D-allulose-3-epimerases described above (also referred to as "mutant D-allulose-3-epimerases").

[0035] As further specific examples, the amino acid sequences of modified D-allulose-3-epimerases are shown in SEQ ID NOs: 16 (M22K) and 17 (G14R). The nucleotide sequences of DNAs encoding the amino acid sequences shown in SEQ ID NOs: 16 and 17 are shown in SEQ ID NOs: 18 and 19.

[0036] The present technology also provides polypeptides described in any of (1) to (4) below: (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, which contains the amino acid substitutions of G14R and / or M22K; (2) a polypeptide comprising the amino acid sequence described in (1) above, in which one or more amino acid residues have been substituted, added, inserted, or deleted, which has D-allulose-3-epimerase activity, and which retains the amino acid substitutions of G14R and / or M22K; and (3) a polypeptide having 90% or more sequence identity to the amino acid sequence described in (1) above, which has D-allulose-3-epimerase activity, and which retains the amino acid substitutions of G14R and / or M22K. (4) A polypeptide comprising the amino acid substitutions described in any of (1) to (3) above, and further comprising one or more amino acid substitutions selected from the group consisting of A56E, N57H, R58G, and F111W.

[0037] The present technology also provides polypeptides described in any of (5) to (7) below: (5) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, which contains one or more amino acid substitutions selected from the group consisting of M22K, G14R, and F111W, (6) a polypeptide consisting of the amino acid sequence described in (1) above, in which one or more amino acid residues have been substituted, added, inserted, or deleted, which has D-allulose-3-epimerase activity, and which retains one or more amino acid substitutions selected from the group consisting of M22K, G14R, and F111W, and (7) a polypeptide which has 90% or more sequence identity to the amino acid sequence described in (1) above, which has D-allulose-3-epimerase activity, and which retains one or more amino acid substitutions selected from the group consisting of M22K, G14R, and F111W.

[0038] The amino acid substitutions in (5) above include single substitutions of M22K only, G14R only, or F111W only; double substitutions of M22K and G14R, M22K and F111W, or G14R and F111W; and triple substitutions of M22K, G14R, and F111W.

[0039] In the polypeptides (2) and (6) above, the introduced amino acid modification may include only one type of modification from substitution, addition, insertion, and deletion (e.g., substitution only, etc.), or may include two or more types of modifications (e.g., substitution and insertion, etc.). In the polypeptides (2) and (6) above, the number of amino acid differences at any difference site may be one or several, for example, 1 to 80, preferably 1 to 70, 1 to 60, 1 to 50, 1 to 40, or 1 to 30, more preferably 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, or 1 to 4, even more preferably 1 to 3, and particularly preferably 1, 2, or 1. However, the amino acid substitutions in (1) and (5) above are retained.

[0040] Furthermore, in the polypeptides (3) and (7) above, the sequence identity to the amino acid sequences of (1) and (5) above may be 70% or more, but is preferably 80% or more, more preferably 85% or more, 90% or more, 95% or more, 96% or more, even more preferably 97% or more, still more preferably 98% or more, and particularly preferably 99% or more.

[0041] Here, in the polypeptides (3) and (7) above, the sequence identity to each of the amino acid sequences (1) and (5) above is the sequence identity calculated by comparing the polypeptides with the amino acid sequences (1) and (5) above. Furthermore, "sequence identity" refers to the amino acid sequence identity value obtained by the bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, pp. 247-250, 1999) of BLASTPACKAGE [sgi32 bit edition, Version 2.0.12; available from the National Center for Biotechnology Information (NCBI)]. The parameters are set as follows: Gap insertion cost value: 11, Gap extension cost value: 1.

[0042] The polypeptides (2) and (6) above, and (3) and (7) above are substantially identical to the polypeptides (1) and (5) above.

[0043] One or more selected from the group consisting of M22K, G14R, A56E, N57H, R58G, and F111W may be part of a larger protein (e.g., a fusion protein, etc.). Examples of sequences added to fusion proteins include sequences that aid in purification, such as multiple histidine residues, and additional sequences that ensure stability during recombinant production.

[0044] In the polypeptides (2) and (6) and (3) and (7) above, when an amino acid substitution is introduced into the polypeptides (1) and (5) above, a preferred embodiment of the amino acid substitution to be introduced is a conservative substitution. That is, examples of the substitution in the polypeptides (2) and (6) and (3) and (7) above include substitution with another nonpolar amino acid if the amino acid before substitution is a nonpolar amino acid, substitution with another uncharged amino acid if the amino acid before substitution is an uncharged amino acid, substitution with another acidic amino acid if the amino acid before substitution is an acidic amino acid, and substitution with another basic amino acid if the amino acid before substitution is a basic amino acid.

[0045] In the polypeptides (2) and (6) and (3) and (7) above, "having D-allulose-3-epimerase activity" means that D-allulose-3-epimerase activity can be detected when measured using the "D-allulose-3-epimerase activity measurement method" in the Examples described below, and preferably means that the D-allulose-3-epimerase activity is equivalent to that of the polypeptides (1) and (5) above; specifically, when the D-allulose-3-epimerase activity of the polypeptides (1) and (5) above is taken as 1, the relative activity is approximately 0.8 to 1.2.

[0046] The above polypeptides (1) to (7) are referred to as D-allulose-3-epimerase derived from Asaia clungsapensis as long as they have D-allulose-3-epimerase activity.

[0047] The present technology also provides DNAs shown in any of the following (1) to (3): (1) DNA encoding a polypeptide set forth in either SEQ ID NO: 16 or 17, (2) a polynucleotide consisting of a nucleotide sequence set forth in either SEQ ID NO: 18 or 19, and (3) a DNA consisting of a sequence equivalent to the nucleotide sequence set forth in either SEQ ID NO: 18 or 19, and encoding a polypeptide having D-allulose-3-epimerase activity.

[0048] Examples of DNA base sequences encoding the amino acid sequence shown in SEQ ID NO: 1 include the base sequences shown in SEQ ID NO: 2 and SEQ ID NO: 15. The base sequence shown in SEQ ID NO: 15 is the base sequence of DNA encoding wild-type D-allulose-3-epimerase derived from Asaia kluangsapensis, and the base sequence shown in SEQ ID NO: 2 is the base sequence of the base sequence shown in SEQ ID NO: 15 modified with codons suitable for Escherichia coli. Therefore, DNA according to the present technology can be appropriately designed by those skilled in the art using the base sequence shown in SEQ ID NO: 2 as a reference sequence. The base sequences shown in SEQ ID NOs: 18 and 19 are base sequences modified with codons suitable for Escherichia coli.

[0049] The DNA encoding D-allulose-3-epimerase is not limited to the above sequence, and may be DNA consisting of a sequence equivalent to the above sequence. Here, an equivalent sequence means DNA having a base sequence that is 70% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, 95% or more, 96% or more, 97% or more, even more preferably 98% or more, and particularly preferably 99% or more homologous to the base sequence of the DNA shown in (1) or (2) above, as long as it encodes a polypeptide having D-allulose-3-epimerase activity.

[0050] Here, DNA "homology" is calculated using publicly available or commercially available software with an algorithm for comparing a reference sequence with a query sequence. Specifically, BLAST, FASTA, or GENETYX (Software Development Co., Ltd.) can be used, and these may be used with default parameters.

[0051] Furthermore, DNA encoding D-allulose-3-epimerase also includes base sequences in which several bases have been mutated by substitution, addition, insertion, or deletion corresponding to the substitution, addition, insertion, or deletion of the amino acid sequence described above, as long as the sequence encodes a polypeptide having D-allulose-3-epimerase activity. Furthermore, base sequences complementary to DNA consisting of a base sequence encoding the above-described polypeptide are also equivalent sequences, and DNA that hybridizes with DNA consisting of such base sequence under stringent conditions is also included in DNA encoding D-allulose-3-epimerase, as long as the DNA encodes a polypeptide having D-allulose-3-epimerase activity. Hybridization methods can be performed according to conventional methods. The same applies hereinafter.

[0052] DNA encoding D-allulose-3-epimerase can also be isolated from a microorganism that produces the above-mentioned predetermined polypeptide. For example, the target DNA can be isolated from the genome of the microorganism by PCR or hybridization using the genomic DNA of Asaia kulungsapensis as a template and primers or probes designed from known amino acid sequence information taking gene degeneracy into consideration, or primers or probes designed based on known nucleotide sequence information.

[0053] DNA encoding D-allulose-3-epimerase includes various types of DNA resulting from codon degeneracy. Various types of DNA encoding the same amino acid sequence can be artificially produced easily using known genetic engineering techniques. For example, in the production of a protein by genetic engineering, if the codons used in the original gene encoding the target protein are used infrequently in the host, the expression level of the protein may be low. In such cases, high expression of the target protein can be achieved by optimizing the codon usage frequency for the host without changing the encoded amino acid sequence.

[0054] Methods for introducing mutations into genes and artificially modifying amino acid sequences include known techniques such as the Kunkel method and the Gapped duplex method, and mutation introduction kits using site-directed mutagenesis, such as QuikChange Site-Directed Mutagenesis Kit (Stratagene), GeneArt Site-Directed Mutagenesis PLUS System (Invitrogen), and TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, PrimeSTAR Mutagenesis Basal Kit, etc.: Takara Bio Inc.). The base sequence of the DNA and whether the obtained DNA encodes the desired polypeptide can be confirmed by conventional methods.

[0055] In the present technology, an expression cassette or recombinant vector containing DNA encoding the above-mentioned specific polypeptide (hereinafter also referred to as the "expression cassette according to the present technology" or the "recombinant vector according to the present technology") can be obtained by linking a promoter and a terminator to DNA encoding D-allulose-3-epimerase, or by inserting the expression cassette according to the present technology or DNA encoding D-allulose-3-epimerase according to the present technology into an expression vector.

[0056] The expression cassette or recombinant vector according to the present technology may contain, as control elements, a promoter and a terminator, as well as transcription elements such as an enhancer, a CCAAT box, a TATA box, or an SPI site, as necessary. These control elements may be operably linked to the DNA according to the present technology. "Operably linked" means that various control elements that regulate the DNA according to the present technology are linked to the DNA of the present invention in a state that allows them to operate in a host cell.

[0057] Regarding the recombinant vector according to the present technology, an expression vector constructed for genetic recombination from a phage, plasmid, or virus capable of autonomously replicating in a host is suitable. Such expression vectors are known, and examples of commercially available expression vectors include pQE-based vectors (manufactured by Qiagen Inc.), pDR540, pRIT2T (manufactured by GE Healthcare Biosciences), and pET-based vectors (manufactured by Merck Ltd.). The expression vector may be used in an appropriate combination with the host cell. For example, when Escherichia coli is used as the host cell, examples include a combination of a pET-based vector and a DH5α Escherichia coli strain, a combination of a pET-based vector and a BL21(DE3) Escherichia coli strain, or a combination of a pDR540 vector and a JM109 Escherichia coli strain.

[0058] Furthermore, a transformant (hereinafter also referred to as "transformant according to the present technology") can be obtained by transforming a host using an expression cassette according to the present technology or a recombinant vector according to the present technology.

[0059] The host used to produce a transformant is not particularly limited as long as it allows gene introduction, the expression cassette or recombinant vector is stable, is capable of autonomous replication, and is capable of expressing the traits of the gene containing the DNA of the present technology, but suitable examples include bacteria belonging to the genus Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus subtilis, the genus Pseudomonas such as Pseudomonas putida, and the genus Asaia such as Asaia krungthepensis; yeast, etc. In addition, animal cells, insect cells, plants, etc. may also be used.

[0060] The transformant according to the present technology can be obtained by introducing the expression cassette or the recombinant vector according to the present technology into a host. The location of introduction of the DNA according to the present technology is not particularly limited as long as the target gene can be expressed, and may be on a plasmid or on the genome. Methods for introducing the expression cassette or the recombinant vector according to the present technology include, for example, a recombinant vector method and a genome editing method. Conditions for introducing the expression cassette or the recombinant vector into a host may be appropriately set depending on the type of host, etc. When the host is a bacterium, examples of the method include a method using competent cells treated with calcium ions and an electroporation method. When the host is a yeast, examples of the method include electroporation, the spheroplast method, and the lithium acetate method. When the host is an animal cell, examples of the method include electroporation, the calcium phosphate method, and the lipofection method. When the host is an insect cell, examples of the method include the calcium phosphate method, the lipofection method, and the electroporation method. When the host is a plant cell, examples of the method include electroporation, Agrobacterium method, particle gun method, PEG method, etc.

[0061] Whether or not the expression cassette according to the present technology or the recombinant vector according to the present technology has been incorporated into a host can be confirmed by PCR, Southern hybridization, Northern hybridization, etc. When confirming whether or not the expression cassette according to the present technology or the recombinant vector according to the present technology has been incorporated into a host by PCR, for example, genomic DNA, the expression cassette, or the recombinant vector may be isolated and purified from the transformant.

[0062] For example, when the host is a bacterium, the isolation and purification of an expression cassette or recombinant vector is carried out using a lysate obtained by lysing the bacteria. Lysis can be achieved, for example, by treatment with a lytic enzyme such as lysozyme, optionally in combination with a protease, other enzymes, and a surfactant such as sodium lauryl sulfate (SDS). Physical disruption methods such as freeze-thawing and French press treatment may also be combined. DNA can be isolated and purified from the lysate by, for example, deproteinization using phenol treatment and protease treatment, ribonuclease treatment, alcohol precipitation, and an appropriate combination of commercially available kits.

[0063] DNA can be cleaved using conventional methods, for example, restriction enzyme treatment. For example, a type II restriction enzyme that acts on a specific nucleotide sequence can be used as the restriction enzyme. The DNA can be linked to an expression cassette or expression vector using, for example, DNA ligase. Then, PCR is performed using the separated and purified DNA as a template and primers specific to the DNA of the present technology. The PCR amplification product can be subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., and stained with ethidium bromide and SYBR Green solution, etc., and the amplification product can be detected as a band to confirm transformation.

[0064] Alternatively, PCR may be performed using primers pre-labeled with a fluorescent dye or the like to detect the amplified product. Furthermore, a method may be employed in which the amplified product is bound to a solid phase such as a microplate and the amplified product is confirmed by fluorescence, enzyme reaction, or the like.

[0065] Furthermore, the present technology encompasses a method for producing a polypeptide, which comprises culturing the above-mentioned transformant.

[0066] The culture conditions for the step of culturing the transformant may be appropriately set taking into consideration the nutritional and physiological properties of the transformant, but liquid culture is preferred. Furthermore, for industrial production, aeration and agitation culture is preferably employed. The nutrient sources used in the medium are those required for the growth of the transformant. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, molasses, and pyruvic acid. The nitrogen source may be any assimilable nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolysate, and alkaline extract of soybean meal. In addition to carbon and nitrogen sources, salts of phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, zinc, and the like; specific amino acids; and specific vitamins may also be used as needed.

[0067] The culture temperature is appropriately set within a range in which the transformant can grow and produce the desired polypeptide. For example, the culture temperature is preferably about 15°C to 37°C. The culture may be completed at an appropriate time when the polypeptide reaches its maximum yield, and the culture time is usually about 12 to 48 hours.

[0068] After culturing the transformant, the culture medium is subjected to centrifugation or other methods, and the culture supernatant and / or bacterial cells are recovered. The bacterial cells are then solubilized by mechanical methods such as ultrasonication or French press treatment, or by treatment with a lytic enzyme such as lysozyme, and optionally with an enzyme such as protease or a surfactant such as sodium lauryl sulfate (SDS), to obtain a water-soluble fraction containing the desired oxygenase. Furthermore, by selecting an appropriate expression cassette or expression vector and host, the expressed polypeptide can be secreted into the culture medium.

[0069] The water-soluble fraction containing the polypeptide obtained as described above may be subjected to purification treatment as is, or the polypeptide in the water-soluble fraction may be concentrated and then subjected to purification treatment. Concentration can be carried out, for example, by vacuum concentration, membrane concentration, salting out treatment, fractional precipitation using a hydrophilic organic solvent (e.g., methanol, ethanol, acetone, etc.), etc. Purification can also be carried out by an appropriate combination of methods such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography. The purified polypeptide of interest may be powdered, if necessary, by freeze-drying, vacuum drying, spray drying, etc.

[0070] Furthermore, modifications such as the addition of sugar chains and / or lipids, or N-terminal or C-terminal processing may be performed. Such modifications can facilitate the extraction and purification of recombinant proteins, or can impart biological functions.

[0071] When Asaia clungsapensis has been caused to produce the D-allulose-3-epimerase represented by the above-mentioned predetermined polypeptide due to mutation, the D-allulose-3-epimerase of the present invention can be produced by culturing the Asaia clungsapensis, recovering it from the culture medium or bacterial cells, and purifying it.

[0072] The content of D-allulose-3-epimerase in the allulose extender according to the present technology can be freely set as long as it does not impair the effects of the present technology. The lower limit of D-allulose-3-epimerase can be set, for example, to 0.0003 U or more per 1 g of fructose used in the production of allulose-containing foods and beverages. From the viewpoint of further enhancing the allulose-increasing effect, it can be set to preferably 0.0015 U or more, more preferably 0.003 U or more, 0.03 U or more, 0.3 U or more, 1 U or more, 10 U or more, 20 U or more, 30 U or more, 40 U or more, 50 U or more, 60 U or more, 70 U or more, even more preferably 70 U or more, and even more preferably 140 U or more.

[0073] The upper limit of the content of D-allulose-3-epimerase in the allulose extender according to the present technology is not particularly limited as long as it does not impair the effects of the present technology, but can be set to, for example, 10,000 U or less, 9,000 U or less, 8,000 U or less, 7,000 U or less, 6,000 U or less, 5,000 U or less, 4,000 U or less, 3,000 U or less, 2,000 U or less, 1,000 U or less, 900 U or less, 800 U or less, 700 U or less, 600 U or less, 500 U or less, 400 U or less, 315 U or less, 300 U or less, 250 U or less, 200 U or less, or 180 U or less per 1 g of fructose used in the production of allulose-containing foods and beverages.

[0074] In this technology, the activity of D-allulose-3-epimerase was measured in 100 mM Tris-HCl buffer (1 mM CoCl ２ ) When reacted with a reaction solution containing pH 7.5 and 50 (v / w)% fructose at 50°C for 1 hour, the amount of enzyme that produces 1 mmol of allulose per minute is defined as 1 unit (1 U).

[0075] (2) Glucosyltransferase The allulose extender according to the present technology preferably further contains glucosyltransferase. By further including glucosyltransferase, the allulose increase effect can be further expected.

[0076] The glucosyltransferase that can be used in the present technology is an enzyme that hydrolyzes sucrose to produce one or more carbohydrates selected from α-1,3 glucan and leucrose. The α-1,3 glucans that can be produced by the glucosyltransferase of the present technology include insoluble α-1,3 glucans and soluble α-1,3 glucans. Furthermore, the insoluble α-1,3 glucans that can be produced by the glucosyltransferase of the present technology include saccharides with seven or more sugars. The soluble α-1,3 glucans that can be produced by the glucosyltransferase of the present technology include nigerooligosaccharides such as nigerose, nigerotriose, nigerotetraose, nigeropentaose, and nigerohexaose. The glucosyltransferase that can be used in the present technology may also have other functions as long as it has glucosyltransferase activity.

[0077] The origin of the glucosyltransferase that can be used in the present technology is not particularly limited, and examples thereof include glucosyltransferases derived from bacteria (e.g., the genus Streptococcus), filamentous fungi (e.g., the genus Aspergillus), etc. Preferably, glucosyltransferases derived from microorganisms of the genus Streptococcus are used, and more preferably, glucosyltransferases derived from Streptococcus thermophilus are used.

[0078] Here, "glucosyltransferase derived from Streptococcus thermophilus" means a glucosyltransferase produced by a microorganism classified as Streptococcus thermophilus (whether a wild-type strain or a mutant strain), or a glucosyltransferase obtained by genetic engineering techniques using a glucosyltransferase gene. Therefore, a recombinant produced by a host microorganism into which a glucosyltransferase gene obtained from Streptococcus thermophilus (or a gene obtained by modifying said gene) has been introduced also falls under the category of "glucosyltransferase derived from Streptococcus thermophilus."

[0079] The strain of Streptococcus thermophilus is not particularly limited as long as it does not impair the action and effect of the present technology, and strains that are generally available from biological resource institutions and the like can be used.

[0080] In the present technology, if necessary, microorganisms that produce enzymes that can be used in the present technology may be cultured. Examples of microorganisms that produce glucosyltransferase include Streptococcus microorganisms, and examples of Streptococcus microorganisms include Streptococcus thermophilus. Microorganisms that can be used may be wild-type strains or mutant strains (e.g., mutant strains induced by ultraviolet irradiation, etc.).

[0081] The culture conditions in the culture step can be appropriately set taking into consideration the properties of the microorganism to be cultured. A solid medium or a liquid medium can be used for culturing the microorganism. In addition, for industrial production, aeration and agitation culture can also be used.

[0082] As the nutrient source for the medium, substances required for the growth of microorganisms can be used as appropriate. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, molasses, or pyruvic acid. The nitrogen source may be any assimilable nitrogen compound, such as peptone, meat extract, yeast extract, casein hydrolysate, or alkaline extract of soybean meal. In addition to carbon and nitrogen sources, salts of phosphate, carbonate, sulfate, magnesium, calcium, potassium, iron, manganese, zinc, or the like; specific amino acids; specific vitamins, or the like, may also be used as needed.

[0083] The culture temperature may be appropriately set within a range in which the microorganism to be cultured can grow and the microorganism can produce the enzyme that can be used in the present technology. The culture temperature may be set, for example, to about 15°C to 37°C. The culture time may be, for example, about 12 hours to 48 hours. The culture step may be terminated, for example, when the enzyme that can be used in the present technology reaches its maximum yield.

[0084] The glucosyltransferase usable in the present technology can be prepared from the culture medium of the microorganism from which the glucosyltransferase is derived. Specific preparation methods include recovering the glucosyltransferase from the culture medium or cells of the microorganism. For example, when a glucosyltransferase-secreting microorganism is used, the cells can be recovered from the culture medium, if necessary, by filtration, centrifugation, or the like, and the enzyme can then be separated and / or purified. When a glucosyltransferase-nonsecreting microorganism is used, the cells can be recovered from the culture medium, if necessary, and then disrupted by pressure treatment, ultrasonication, or the like to extract the enzyme, after which the enzyme can be separated and / or purified. The enzyme can be separated and / or purified by any known protein separation and / or purification method, without any particular limitation. Examples of the enzyme separation and / or purification method include centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins, etc. The separated and / or purified enzyme can be powdered by drying methods such as lyophilization and vacuum drying, or by using appropriate excipients and / or drying aids in the drying methods. The isolated and / or purified enzyme can also be liquefied by adding an appropriate additive and sterilizing by filtration.

[0085] In the present technology, specific examples of glucosyltransferases include the glucosyltransferases derived from Streptococcus thermophilus shown below. For example, a glucosyltransferase derived from Streptococcus thermophilus containing a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 20 is included. An amino acid sequence including a signal sequence and a pro-sequence is shown in SEQ ID NO: 21. Another specific example is a glucosyltransferase derived from Streptococcus thermophilus containing a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 22. An amino acid sequence including a signal sequence and a pro-sequence is shown in SEQ ID NO: 23.

[0086] The glucosyltransferase that can be used in the present technology may be not only the polypeptide shown in (1) below, but also glucosyltransferases including the polypeptides shown in (2) to (4) below. Furthermore, the glucosyltransferase that can be used in the present technology may be an artificial protein artificially designed based on the amino acid sequences shown in (1) to (4) below. (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:20 or SEQ ID NO:22; (2) A polypeptide having glucosyltransferase activity, in which one or more amino acid residues have been substituted, added, inserted, deleted, or inverted in the amino acid sequence shown in SEQ ID NO:20 or SEQ ID NO:22; (3) A polypeptide having glucosyltransferase activity, in which the amino acid sequence shown in SEQ ID NO:20 has 93% or more sequence identity to the amino acid sequence shown in SEQ ID NO:20; and (4) A polypeptide having glucosyltransferase activity, in which the amino acid sequence shown in SEQ ID NO:22 has 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO:22.

[0087] The lower limit of the content of glucosyltransferase in the allulose extender of the present technology can be freely set as long as it does not impair the effect of the present technology. The content of glucosyltransferase can be set to, for example, 0.0001U or more per 1g of fructose used in the production of allulose-containing foods and beverages. From the viewpoint of further enhancing the allulose-increasing effect, it can be set to preferably 0.001U or more, more preferably 0.01U or more, 0.03U or more, 0.05U or more, 0.1U or more, 0.5U or more, 1U or more, 5U or more, even more preferably 7.5U or more, and even more preferably 10U or more.

[0088] The upper limit of the glucosyltransferase content in the allulose extender of the present technology is not particularly limited as long as it does not impair the effects of the present technology, but can be set to, for example, 500 U or less, 400 U or less, 300 U or less, 200 U or less, 100 U or less, or 50 U or less per 1 g of fructose used in the production of allulose-containing foods and beverages.

[0089] In this technology, the activity of glucosyltransferase is defined as one unit (1 U) of the enzyme amount that hydrolyzes 1 μmol of sucrose per minute when treated at 30°C using 50 mM sucrose solution (50 mM potassium phosphate buffer, pH 7.0) as a substrate.

[0090] (3) Other Components The allulose bulking agent of the present technology can be used in combination with other components as long as it does not impair the action or effect of the present technology. Examples of other components that can be used include excipients, pH adjusters, colorants, flavoring agents, disintegrants, lubricants, stabilizers, enzymes, and other components that are commonly used in formulations. Furthermore, components with known or future functions can also be used in combination as appropriate depending on the purpose.

[0091] 2. Allulose-Containing Food and Drink The allulose-containing food and drink according to the present technology will be described in detail below.

[0092] (1) Raw materials Raw materials that can be used for the allulose-containing food and drink or fructose-containing food and drink according to the present technology are not particularly limited in origin, type, etc., as long as they do not impair the effects of the present technology, and can be freely selected depending on the target food and drink, but raw materials containing fructose (fructose-containing raw materials) are preferred.Specific examples include fruit juices such as grapes, peaches, bananas, apples, strawberries, melons, mangoes, plums, cherries, oranges, Satsuma mandarins, lemons, grapefruits, limes, mandarins, yuzu, tangerines, temple oranges, tangelos, calamansi, persimmons, blueberries, pineapples, papayas, lychees, acerola, pears, pears, apricots, guavas, prunes, and apricots. Other examples include vegetable juices from carrots, tomatoes, bell peppers, paprika, Japanese mustard spinach, kale, broccoli, spinach, asparagus, daikon radish, Chinese cabbage, celery, cabbage, beets, onions, lettuce, parsley, watercress, pumpkin, watermelon, corn, etc. The fruit juice and / or vegetable juice may be used alone or in combination of two or more.

[0093] (2) Fructose-Containing Foods and Drinks In the present technology, fructose-containing foods and drinks include those produced by processing or cooking the above-mentioned raw materials. Examples of fructose-containing foods and drinks include one or more selected from the group consisting of fruit juice drinks, vegetable juice drinks, and mixed juice drinks. Preferred examples include fruit juice drinks and vegetable juice drinks, and more preferred examples include apple juice drinks and carrot juice drinks.

[0094] In this technology, raw materials or fructose-containing food or beverage are the target of the allulose extender, and as described below, the allulose extender of this technology reduces the amount of fructose in the raw materials or fructose-containing food or beverage and increases the amount of allulose.

[0095] (3) Allulose-Containing Food and Drink The allulose-containing food and drink according to the present technology is a food and drink produced by using an allulose extender in the above-mentioned raw material or fructose-containing food and drink.

[0096] The amount of sugars contained in the allulose-containing food and drink of the present technology is not particularly limited, but it is preferable that the amount of fructose in the raw material or the raw material of the fructose-containing food and drink is reduced and the amount of allulose is increased.The content of fructose (also called "fruit sugar") contained in the raw material or fructose-containing food and drink that can be used in the present technology can be freely set as long as it does not achieve the function or effect of the present technology.The content of fructose is, for example, 1 wt% or more, 2 wt% or more, 3 wt% or more, 4 wt% or more, 5 wt% or more, or 6 wt% or more.

[0097] The sugar composition of allulose contained in the allulose-containing food and drink according to the present technology is, for example, 1% or more, preferably 1.5% or more, more preferably 5% or more, and even more preferably 7.5% or more. The upper limit of the sugar composition of allulose contained in the allulose-containing food and drink according to the present technology is not particularly limited, and is, for example, 30% or less, 25% or less, 20% or less, or 15% or less.

[0098] The sugar composition of fructose contained in the allulose-containing food or drink according to the present technology is, for example, 80% or less, preferably 70% or less, more preferably 65% ​​or less, and even more preferably 50% or less. The lower limit of the sugar composition of fructose contained in the allulose-containing food or drink according to the present technology is not particularly limited, and is, for example, 20% or more, 30% or more, or 40% or more.

[0099] Specific examples of the allulose-containing food and drink according to the present technology include one or more selected from the group consisting of fruit juice drinks, vegetable juice drinks, and mixed juice drinks. Fruit juice drinks and vegetable juice drinks are preferred, and apple juice drinks and carrot juice drinks are more preferred.

[0100] (4) Others The allulose bulking agent according to the present technology can also be used in plant-derived molasses. Examples of plant-derived molasses raw materials include honey, agave syrup, and maple syrup.

[0101] In the present technology, the properties of the raw material or fructose-containing food or drink when subjected to various enzyme treatments are not particularly limited as long as the effects of the present technology are not impaired, but are preferably in the form of a liquid or paste, more preferably in the form of a liquid. Furthermore, when a raw material having a skin is used, either peeled or unpeeled raw materials can be used.

[0102] 3. Method for producing allulose-containing foods and beverages, and method for increasing allulose in fructose-containing foods and beverages The method for producing allulose-containing foods and beverages and method for increasing allulose in fructose-containing foods and beverages according to the present technology include a step of treating the raw material or fructose-containing foods and beverages with D-allulose-3-epimerase (also referred to as the "enzyme action step (I)"). Furthermore, the method for producing allulose-containing foods and beverages and method for increasing allulose in fructose-containing foods and beverages according to the present technology may further include a step of treating the raw material or fructose-containing foods and beverages with glucosyltransferase (also referred to as the "enzyme action step (II)"). Furthermore, depending on the type of raw material or fructose-containing foods and beverages, general food and beverage production processes and recovery processes can be performed before, after, or simultaneously with each step, as long as they do not impair the effects of the present technology. Each step is described in detail below.

[0103] (1) Enzyme action step (I) The enzyme action step (I) is a step in which D-allulose-3-epimerase is allowed to act on the raw material or a fructose-containing food or drink (including the step of allowing any of the polypeptides shown in (1) to (3) above to act on fructose). If necessary, other enzymes may also be allowed to act. These enzymes may be used singly or in combination of two or more. When using a combination of two or more, the two or more enzymes may be added simultaneously or separately to the raw material or fructose-containing food or drink. The order in which two or more enzymes are added separately is not particularly limited. The details and amounts of the enzymes to be added are the same as those of the enzymes used in "1. Allulose extender" above, and therefore will not be described here.

[0104] The specific method of adding each enzyme is not particularly limited, and as long as the enzyme can be added to the raw material or the fructose-containing food or drink, it can be added by any method and at any timing. Furthermore, each enzyme can be added in two or more separate portions.

[0105] The various conditions for the enzyme action step (I) can be freely set as long as they do not impair the effects of the present technology. For example, the pH, temperature, action time, etc. can be set depending on the physicochemical properties of the enzyme used, such as the optimal pH, stable pH range, optimal temperature, and temperature stability. The optimal reaction conditions can be determined through preliminary experiments. Examples of conditions for enzyme action are given below.

[0106] When D-allulose-3-epimerase is added as an enzyme to a raw material or a fructose-containing food or drink, the pH can be set, for example, to 2.0 to 13.0, preferably 2.5 to 12.0, and more preferably 3.0 to 8.0. The temperature can be set, for example, to 20°C to 90°C, preferably 30°C to 80°C, and more preferably 40°C to 70°C. The reaction time can be set, for example, to 10 minutes to 6 hours, preferably 30 minutes to 4 hours, and more preferably 1 hour to 3 hours.

[0107] (2) Enzyme action step (II) The enzyme action step (II) is a step of allowing glucosyltransferase to act on the raw material or fructose-containing food or drink. The details and amount of the enzyme to be added are the same as those of the enzyme used in "1. Allulose extender" above, so a description thereof will be omitted here.

[0108] The specific method of adding glucosyltransferase is not particularly limited, and as long as the enzyme can be added to the raw material or the fructose-containing food or drink, it can be added by any method and at any timing. In addition, glucosyltransferase can be added in two or more separate portions.

[0109] The various conditions for the enzymatic reaction step (II) can be freely set as long as they do not impair the effects of the present technology. For example, the pH, temperature, reaction time, etc. can be set according to the physicochemical properties of the enzyme used, such as the optimal pH, stable pH range, optimal temperature, and temperature stability. The optimal reaction conditions can be determined through preliminary experiments. Below are examples of conditions for the use of glucosyltransferase.

[0110] When glucosyltransferase is added as an enzyme to a raw material or a fructose-containing food or drink, the pH can be set, for example, to 2.0 to 9.0, preferably 2.5 to 8.0, and more preferably 3.0 to 6.0. The temperature can be set, for example, to 10°C to 70°C, preferably 20°C to 50°C, and more preferably 25°C to 45°C. The reaction time can be set, for example, to 15 minutes to 120 hours, preferably 30 minutes to 72 hours, and more preferably 2 hours to 48 hours.

[0111] (3) Raw Material Preparation Step The raw material preparation step is a step of preparing the enzymes (D-allulose-3-epimerase and / or glucosyltransferase), raw materials (including fructose) used in the production of food and beverages, etc. The raw materials may be prepared in the form of a composition mixed with other ingredients, as long as the action and effect of the present technology are not impaired.

[0112] (4) Enzyme deactivation step The enzyme deactivation step is a step of deactivating the enzyme after at least the above-mentioned enzymatic action step (I). When the enzymatic action step (II) has been performed, the enzyme can also be deactivated after the enzymatic action step (II).

[0113] The enzyme deactivation step is not an essential step and can be carried out as needed. Furthermore, when multiple enzymes are used in the enzyme action step (I) or enzyme action step (II), the enzyme deactivation step can also be carried out between the actions of the enzymes. Furthermore, for example, when multiple enzymes are allowed to act in the enzyme action step (I) or enzyme action step (II), the enzyme deactivation step can also be carried out midway through the enzyme action step (I) or enzyme action step (II). Furthermore, the enzyme deactivation step can also be carried out between the enzyme action step (I) and the enzyme action step (II).

[0114] The method for inactivating the enzyme is not particularly limited, and a general inactivation method may be used alone or in combination of two or more depending on the properties of the enzyme to be inactivated. For example, a method of inactivating the enzyme by heating to a temperature equal to or higher than the enzyme inactivation temperature may be used.

[0115] (5) Recovery step The recovery step is a step of recovering the allulose-containing food or drink obtained through the enzyme action step (I) and, if necessary, other steps. The specific recovery method may be one of the recovery methods commonly used in the production of allulose-containing food or drink, or a combination of two or more methods, depending on the state and type of the obtained allulose-containing food or drink.

[0116] The method according to the present technology described above can be embodied as comprising the following steps A to D. A: A step of preparing a raw material or a fructose-containing food or drink B: A step of allowing D-allulose-3-epimerase to act on the prepared raw material or fructose-containing food or drink C: A step of allowing glucosyltransferase to further act D: A step of inactivating the enzyme E: A step of recovering the produced allulose-containing food or drink

[0117] Steps C and D are not essential and can be performed as needed. Furthermore, step D can be performed multiple times as needed after step B, during step B, after step C, during step C, during step E, or after step E. Furthermore, in step D, the enzyme can also be inactivated by heating or the like in the production process of the allulose-containing food or drink. In addition, when multiple enzymes are used, enzyme inactivation can also be performed between the reactions of the enzymes (for example, between step B and step C). For example, when multiple enzymes are used in step B and / or step C, step D can also be appropriately performed during step B and / or step C. Furthermore, step B and step C may be performed simultaneously, step C may be performed after step B, or step B may be performed after step C.

[0118] The present technology can employ the following configurations. [1] An allulose extender comprising D-allulose-3-epimerase. [2] The allulose extender according to [1], wherein the D-allulose-3-epimerase is derived from Asaia krungthepensis. [3] The allulose extender according to [1] or [2], further comprising glucosyltransferase. [4] A food or drink in which the allulose extender according to any one of [1] to [3] is used. [5] A method for producing an allulose-containing food or drink, comprising a step of allowing D-allulose-3-epimerase to act on a raw material or a fructose-containing food or drink. [6] A method for producing an allulose-containing food or drink according to [5], further comprising a step of allowing glucosyltransferase to act on a raw material or a fructose-containing food or drink. [7] A method for increasing the allulose content of a fructose-containing food or drink, comprising the step of treating a raw material or a fructose-containing food or drink with D-allulose-3-epimerase. [8] The method for increasing the allulose content of a fructose-containing food or drink according to [7], further comprising the step of treating a raw material or a fructose-containing food or drink with glucosyltransferase. [9] A polypeptide shown in any of the following (1) to (3): (1) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, which contains the amino acid substitution of G14R and / or M22K, (2) a polypeptide comprising the amino acid sequence of (1) above, in which one or more amino acid residues have been substituted, added, inserted or deleted, and which has D-allulose-3-epimerase activity and retains the amino acid substitution of G14R and / or M22K, and (3) a polypeptide which has 90% or more sequence identity to the amino acid sequence of (1) above, has D-allulose-3-epimerase activity and retains the amino acid substitution of G14R and / or M22K.

[10] The polypeptide of [9], further comprising one or more amino acid substitutions selected from the group consisting of A56E, N57H, R58G, and F111W.

[11] A DNA shown in any one of (1) to (3) below: (1) a DNA encoding the polypeptide of [9], (2) a polynucleotide consisting of the nucleotide sequence shown in either SEQ ID NO: 18 or 19, (3) a DNA encoding a polypeptide having D-allulose-3-epimerase activity, which consists of a sequence equivalent to the nucleotide sequence shown in either SEQ ID NO: 18 or 19.

[12] An expression cassette or recombinant vector comprising the DNA of

[11] .

[13] A transformant obtained by transforming a host with the expression cassette or recombinant vector of

[12] .

[14] A method for producing the polypeptide of [9], which comprises a step of culturing the transformant of

[13] .

[15] A method for producing allulose, which comprises a step of allowing the polypeptide of [9] to act on fructose.

[0119] The present technology will be described in more detail below based on examples. Note that the examples described below are examples of typical examples of the present technology, and the scope of the present technology should not be construed as being narrow.

[0120] <Experimental Example 1> (1) Raw Materials The raw materials and enzymes used in Experimental Example 1 are shown in Table 1 below.

[0121]

[0122] (2) Method for Measuring D-Allulose-3-Epimerase Activity: D-Allulose-3-Epimerase was assayed in 111 mM Tris-HCl buffer (1.11 mM CoCl) containing 50 (v / w)% fructose. ２ To 90 mL of a 10 mL sample solution containing DAE (pH 7.5), 10 mL of the sample solution was added, and the reaction solution was treated at 50° C. for 1 hour, and then boiled at 100° C. for 5 minutes. After cooling, fructose and allulose were detected by HPLC, and the enzyme activity was calculated by defining the amount of enzyme that produces 1 mmol of allulose per minute as 1 U.

[0123] (3) Experimental Examples (3-1) Methods To prepare apple juice beverages with various pH values ​​(pH 4.0, 5.0, 6.0, and 7.0), an apple juice beverage (Brix 11, pH 3.75, manufactured by Kagome Co., Ltd.) was used as a base. 1N aqueous sodium hydroxide solution was added to the apple juice beverage to prepare apple juice beverages with various pH values ​​(pH 4.0, 5.0, 6.0, and 7.0). D-allulose-3-epimerase (having the amino acid sequence set forth in SEQ ID NO: 1) was added at 156 U per 1 g of fructose to each apple juice (pH 4.0, 5.0, 6.0, and 7.0), and the enzyme reaction was carried out at 50°C for 2 hours. After the enzyme reaction, the apple juice beverage was heated at 100°C for 10 minutes to inactivate the enzyme, thereby preparing each enzyme-treated apple juice beverage.

[0124] (3-2) Analysis of Sugar Composition The sugar contents of each of the enzyme-treated apple juice beverages prepared in (3-1) above and the untreated apple juice beverage (control; Control Example 1) were measured using the following procedure. One mL of the reaction solution (centrifugal supernatant) was filtered through a membrane, and the sugar composition was analyzed by high-performance liquid chromatography (hereinafter also referred to as "HPLC") under the conditions shown in Table 2 below. From the obtained sugar composition, the ratios of allulose, fructose, and glucose to the total sugar content (sugar composition) were calculated.

[0125]

[0126] (3-3) Results The results are shown in Table 3 below.

[0127]

[0128] (3-4) Discussion As shown in Table 3 above, comparing the untreated (Control Example 1) and treated with enzymes (Examples 1 to 4), allulose was produced at a pH of 5.0 or higher. In other words, it was confirmed that an apple juice beverage with increased allulose content could be obtained.

[0129] <Experimental Example 2> (1) Raw Materials The raw materials and enzymes used in Experimental Example 2 are shown in Table 4 below.

[0130]

[0131] (2) Method for Measuring D-Allulose-3-Epimerase Activity The enzyme activity was determined in the same manner as in Experimental Example 1 above.

[0132] (3) Glucosyltransferase activity measurement method As a substrate, 1700 μL of 50 mM potassium phosphate buffer (pH 7.0), 100 μL of 1 M sucrose solution (50 mM (1.71%) (17.1 g / L)), and 200 μL of enzyme solution were added to a test tube, mixed, and reacted at 30 ° C. for 1 hour. After the reaction, the amount of free fructose was measured using E-kit Liquid D-glucose / fructose (manufactured by JK International Co., Ltd.), and the activity was calculated as a relative value. When 50 mM sucrose solution (50 mM phosphate buffer (pH 7.0)) was used as a substrate and treated at 30 ° C., the amount of enzyme hydrolyzing 1 μmol of sucrose per minute was defined as 1 U, and the enzyme activity was calculated.

[0133] (4) Experimental Example (4-1) Method Using a carrot juice drink (pH 5.12, manufactured by Kagome Co., Ltd., the same applies below) as a base, 35.3 U of D-allulose-3-epimerase (D-allulose-3-epimerase having the amino acid sequence shown in SEQ ID NO: 1) per 1 g of sucrose, and 12.8 U of glucosyltransferase 1 (glucosyltransferase having the amino acid sequence shown in SEQ ID NO: 20) or glucosyltransferase 2 (glucosyltransferase having the amino acid sequence shown in SEQ ID NO: 22) per 1 g of sucrose were added to the carrot juice drink, and after stirring by inversion, the mixture was allowed to react at 30°C for 24 hours.

[0134] (4-2) Analysis of Sugar Composition The sugar contents of each of the enzyme-treated carrot juice beverages prepared in (4-1) above and the non-enzyme-treated carrot juice beverages (controls; Control Examples 2 and 3) were measured by the following procedure. 1 mL of the reaction solution (centrifugation supernatant) was filtered through a membrane, and the sugar composition was analyzed by HPLC under the conditions shown in Table 5 below. From the obtained sugar composition, the ratios of allulose, fructose, and glucose to the total sugar content (sugar composition) were calculated.

[0135]

[0136] (4-3) Results The results are shown in Tables 6 and 7 below.

[0137]

[0138]

[0139] (4-4) Discussion As shown in Tables 6 and 7 above, when comparing the non-enzyme-treated (Control Examples 2 and 3) with the enzymatically treated carrot juice beverages, Examples 5 and 7, which were enzymatically treated with only D-allulose-3-epimerase, yielded carrot juice beverages with increased allulose. On the other hand, although no increase in allulose was confirmed in either Reference Example 1, which was enzymatically treated with only glucosyltransferase 1, or Reference Example 2, which was enzymatically treated with only glucosyltransferase 2, a significant increase in allulose was confirmed in both Examples 6 and 8, which were enzymatically treated with both D-allulose-3-epimerase.

[0140] <Experimental Example 3> (1) Raw Materials The raw materials and enzymes used in Experimental Example 3 are shown in Table 8 below.

[0141]

[0142] (2) Obtaining and Identifying the Enzyme Using the full-length D-allulose-3-epimerase sequence (SEQ ID NO: 2) introduced into the pET24 vector as a template, PCR was performed in a conventional manner to introduce mutations.

[0143] E. coli JM109 was transformed with the resulting amplification product to obtain a gene expression vector. The D-allulose-3-epimerase sequence introduced into the gene expression vector was confirmed. The gene expression vector was introduced into E. coli FBL21(DE3). The transformant was cultured in LB medium at 37°C for 3 hours with shaking. After 20 minutes of shaking culture at 25°C, IPTG (final concentration 1 mM) was added and the culture was continued with shaking at 25°C for 24 hours. The cells were recovered from the culture medium, suspended in 50 mM Tris-HCl buffer (pH 7.5), and disrupted using a bead shocker. The mixture was centrifuged at 15,000 rpm for 10 minutes. The recovered supernatant was used as the enzyme sample.

[0144] (2) Measurement of D-allulose-3-epimerase activity Method The enzyme activity was determined in the same manner as in Experimental Example 1 above.

[0145] (3) Experimental Examples (3-1) Method In order to prepare apple juice beverages with various pH values ​​using an apple juice beverage (Brix 11, pH 3.75, manufactured by Kagome Co., Ltd.; the same applies hereinafter) as a base, 1N aqueous sodium hydroxide solution was added to the apple juice beverage to prepare apple juice beverages with various pH values ​​(pH 4.0, 5.0, 6.0, and 7.0). D-allulose-3-epimerase (Variant 1 (M22K): D-allulose-3-epimerase having the amino acid sequence shown in SEQ ID NO: 16; Variant 2 (G14R): D-allulose-3-epimerase having the amino acid sequence shown in SEQ ID NO: 17; Variant 3 (F111W): D-allulose-3-epimerase having the amino acid sequence shown in SEQ ID NO: 6) was added to each apple juice beverage (pH 4.0, 5.0, 6.0, and 7.0) at a concentration of 156 U per 1 g of fructose, and the enzyme reaction was carried out at 50°C for 2 hours. After the enzyme reaction, the apple juice beverage was centrifuged at 15,000 G for 5 minutes, and each enzyme-treated apple juice beverage was prepared.

[0146] (3-2) Analysis of Sugar Composition The sugar content of each enzyme-treated apple juice beverage prepared in (3-1) above was measured using the following procedure. 1 mL of the reaction solution (centrifugal supernatant) was filtered through a membrane, and the sugar composition was analyzed by HPLC under the conditions shown in Table 9 below. From the obtained sugar composition, the ratio of allulose, fructose, and glucose to the total sugar content (sugar composition) was calculated.

[0147]

[0148] (3-3) Results The results are shown in Tables 10 and 11 below.

[0149]

[0150]

[0151] (3-4) Discussion As shown in Tables 10 and 11 above, when the enzyme treatments (Examples 7 to 10) were compared, allulose was also produced at pH 5.0 or higher in D-allulose-3-epimerase variants 1 to 3. Furthermore, in particular, D-allulose-3-epimerase variants 1 and 2 produced greater amounts of allulose at pH 5.0 or higher. In other words, it was confirmed that an apple juice beverage with increased allulose content could be obtained.

Claims

1. An allulose extender comprising D-allulose-3-epimerase.

2. The allulose extender of claim 1, wherein the D-allulose-3-epimerase is derived from Asaia krungthepensis.

3. The allulose extender of claim 1, further comprising a glucosyltransferase.

4. An allulose-containing food or beverage, which uses an allulose extender described in any one of claims 1 to 3.

5. A method for producing allulose-containing food or beverage, comprising a step of reacting D-allulose-3-epimerase with a raw material or a fructose-containing food or beverage.

6. A method for producing an allulose-containing food or beverage as described in claim 5, further comprising a step of reacting a glucosyltransferase with a raw material or a fructose-containing food or beverage.

7. A method for increasing allulose in fructose-containing food or beverage, comprising a step of treating a raw material or a fructose-containing food or beverage with D-allulose-3-epimerase.

8. A method for increasing the amount of allulose in a fructose-containing food or beverage as described in claim 7, further comprising a step of reacting a glucosyltransferase with the raw material or the fructose-containing food or beverage.

Images