Fructosylated maltitol and method for producing same

Abstract

The purpose of the present invention is to provide a novel fructosylated maltitol and a method for producing the same. According to the present invention, provided is a method for producing fructosylated maltitol, which comprises a step (contact step) in which beta-fructofuranosidase having a beta-fructofuranosidase activity and having a sequence identity of 60% or more to the amino acid sequence represented by SEQ ID NO: 1 is brought into contact with sucrose and maltitol. According to the present invention, it is advantageous in that fructosylated maltitol can be easily and efficiently produced.

Description

[0001] Reference to related applications

[0002] This application claims the benefit of priority from a prior Japanese application, namely, Japanese Patent Application No. 2023-7058 (filing date: January 20, 2023), the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to fructosylated maltitol and a method for producing the same. Background Art

[0004] The intestinal flora significantly impacts human health, and therefore, health-promoting effects can be achieved by improving the balance of microorganisms that make up the intestinal flora. Prebiotics are indigestible food ingredients that selectively promote the proliferation and activity of specific beneficial microorganisms, such as Bifidobacterium, that inhabit the intestinal environment. Unlike probiotics, which promote health through the ingestion of exogenous microorganisms, prebiotics are a more practical and effective method for manipulating the intestinal flora because the target bacteria already coexist in the intestines (Non-Patent Document 1).

[0005] Sugar alcohols are known to be low-calorie sweeteners that are not digested and absorbed but reach the large intestine to be utilized by intestinal bacteria, and are also expected to function as prebiotics. On the other hand, there is a demand for the development of prebiotic raw materials with better functions and physical properties. As a method for fructosylation of sugar alcohols, for example, the use of a yeast derived from Schwann yeast ( Schwanniomyces ) genus (Non-Patent Document 2).

[0006] Prior art literature

[0007] Non-patent literature

[0008] Non-patent literature 1: Gibson GR, et al., J Nutr., 1995; 125(6): 1401-1412.

[0009] Non-patent literature 2: Piedrabuena D et al., Appl Microbiol Biotechnol. 2016; 100(20): 8769-8778. Summary of the Invention

[0010] Problems to be solved by the invention

[0011] The object of the present invention is to provide a novel fructosylated maltitol and a method for producing the same.

[0012] Solutions for solving problems

[0013] The present inventors have found that by using Aspergillus Fijiensis ( Aspergills fijiensis ) can produce fructosylated maltitol by contacting sucrose and maltitol with β-fructofuranosidase. The present inventors have also discovered that the β-fructofuranosidase has substrate specificity for maltitol among sugar alcohols. The present inventors have further discovered that increasing the ratio of maltitol to sucrose during the contacting process increases the amount of fructosylated maltitol produced. The present invention is based on these findings.

[0014] According to the present invention, the following technical solutions are provided.

[0015] [1] A method for producing fructosylated maltitol, comprising contacting a culture of (A) β-fructofuranosidase or (B) a polypeptide comprising a portion thereof or (C) a transformant comprising either or both thereof with sucrose and maltitol (contacting step);

[0016] The (A) β-fructofuranosidase has a sequence identity of more than 60% with the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity.

[0017] The polypeptide (B) comprises an enzymatically active portion of the amino acid sequence of β-fructofuranosidase having the amino acid sequence shown in SEQ ID NO: 1, and has β-fructofuranosidase activity.

[0018] The culture of the transformant (C) is obtained by introducing an expression vector containing a polynucleotide encoding the (A) β-fructofuranosidase or a polynucleotide encoding the (B) polypeptide into a host.

[0019] [2] The production method according to [1] above, wherein the β-fructofuranosidase has substrate specificity for maltitol among sugar alcohols.

[0020] [3] The production method according to [1] or [2] above, wherein the ratio of the amount or concentration of maltitol to sucrose at the start of contact is 1 or more.

[0021] [4] The production method according to any one of [1] to [3] above, wherein the temperature in the contacting step is 40°C to 60°C.

[0022] [5] The production method according to any one of [1] to [4] above, wherein the contacting step is performed for 8 hours or longer.

[0023] [6] The production method according to any one of [1] to [5] above, wherein the host is selected from the group consisting of bacteria, yeast, mold and filamentous fungi.

[0024] [7] The production method according to any one of [1] to [6] above, wherein the β-fructofuranosidase is derived from a microorganism of the genus Aspergillus.

[0025] [8] The production method according to any one of [1] to [7] above, wherein the β-fructofuranosidase is derived from Aspergillus fijiensis.

[0026] [9] An enzyme agent for producing fructosylated maltitol, comprising: a culture of a β-fructofuranosidase or a polypeptide comprising a portion thereof, or a transformant comprising either or both thereof;

[0027] The β-fructofuranosidase has a sequence identity of more than 60% with the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity.

[0028] The polypeptide comprises an enzymatically active portion of the amino acid sequence of β-fructofuranosidase having the amino acid sequence shown in SEQ ID NO: 1, and has β-fructofuranosidase activity.

[0029] The culture of the transformant is a culture of the transformant obtained by introducing an expression vector containing a polynucleotide encoding the β-fructofuranosidase or a polynucleotide encoding the polypeptide into a host.

[0030]

[10] A fructosylated maltitol, which is maltitol to which one or two molecules of fructose are bonded.

[0031]

[11] The fructosylated maltitol according to

[10] above, wherein the maltitol to which one molecule of fructose is bonded is represented by the following formula (I).

[0032]

[12] The fructosylated maltitol according to

[10] above, wherein the maltitol to which two molecules of fructose are bonded is represented by the following formula (II).

[0033]

[13] An enzyme reaction composition comprising: (D) β-fructofuranosidase as an enzyme, or (E) a polypeptide comprising a portion thereof, or (F) a culture of a transformant comprising either or both thereof, and sucrose and maltitol as substrates, wherein the proportion of fructosylated maltitol relative to the total sugars in the composition is 5% or more, and

[0034] The (D) β-fructofuranosidase has a sequence identity of more than 60% with the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity.

[0035] The polypeptide (E) comprises an enzymatically active portion of the amino acid sequence of β-fructofuranosidase having the amino acid sequence shown in SEQ ID NO: 1, and has β-fructofuranosidase activity.

[0036] The culture of the (F) transformant is obtained by introducing an expression vector containing a polynucleotide encoding the (D) β-fructofuranosidase or a polynucleotide encoding the (E) polypeptide into a host.

[0037]

[14] A use of β-fructofuranosidase for producing fructosylated maltitol, wherein the β-fructofuranosidase has a sequence identity of more than 60% with the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity.

[0038] The present invention is advantageous in that fructosylated maltitol can be produced simply and efficiently. Furthermore, the β-fructofuranosidase has substrate specificity for maltitol among sugar alcohols, thereby producing fructosylated maltitol with little production of byproducts, which is also advantageous. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] [ Figure 1 ] Figure 1 A represents the results of HPLC analysis of the reaction solution when maltitol was used as the sugar alcohol (chromatogram). The solid line in the figure represents the substrate being sucrose alone (48 hours), the dashed line represents the substrate being sucrose and maltitol (sucrose + maltitol) (0 hours), and the dotted line represents the substrate being sucrose and maltitol (sucrose + maltitol) (48 hours) (the time in parentheses represents the reaction time). The asterisks in the figure represent maltitol bonded to one molecule of fructose (the asterisk on the left) and maltitol bonded to two molecules of fructose (the asterisk on the right), respectively. Figure 1 B represents the ratio of each sugar to the total sugar in the reaction solution when maltitol is used as the sugar alcohol.

[0040] [ Figure 2 ] Figure 2 A represents the results of HPLC analysis of the reaction solution when lactitol was used as the sugar alcohol (chromatogram). The solid line in the figure represents the substrate being sucrose alone (48 hours), the dashed line represents the substrate being sucrose and lactitol (sucrose + lactitol) (0 hours), and the dotted line represents the substrate being sucrose and lactitol (sucrose + lactitol) (48 hours) (time in parentheses represents reaction time). Figure 2 B represents the ratio of each sugar to the total sugar in the reaction solution when lactitol is used as the sugar alcohol.

[0041] [ Figure 3 ] Figure 3A represents the results of HPLC analysis of the reaction solution when isomalt was used as the sugar alcohol (chromatogram). The solid line in the figure represents the substrate being sucrose alone (48 hours), the dashed line represents the substrate being sucrose and isomalt (sucrose + isomalt) (0 hours), and the dotted line represents the substrate being sucrose and isomalt (sucrose + isomalt) (48 hours) (time in parentheses represents reaction time). Figure 3 B represents the ratio of each sugar to the total sugar in the reaction solution when isomalt is used as the sugar alcohol.

[0042] [ Figure 4 ] Figure 4 A represents the results of HPLC analysis of the reaction solution when xylitol was used as the sugar alcohol (chromatogram). The solid line in the figure represents the substrate being sucrose alone (48 hours), the dashed line represents the substrate being sucrose and xylitol (sucrose + xylitol) (0 hours), and the dotted line represents the substrate being sucrose and xylitol (sucrose + xylitol) (48 hours) (time in parentheses represents reaction time). Figure 4 B represents the ratio of each sugar to the total sugar in the reaction solution when xylitol is used as the sugar alcohol.

[0043] [ Figure 5 ] Figure 5 A represents the ratio of each sugar to the total sugar in the reaction solution when maltitol was used at a ratio of 1:1, 1:2, and 1:3 to sucrose. Figure 5 B represents the ratio of each FOS to the total FOS in the reaction solution when maltitol was used at a ratio of 1:1, 1:2, and 1:3 to sucrose.

[0044] [ Figure 6 ] Figure 6 The graphs show the changes in the ratio of maltitol bound to one molecule of fructose (×) to the total sugars, the ratio of maltitol bound to two molecules of fructose (●) to the total sugars, and the ratio of their sum (◆) to the total sugars at each reaction time when maltitol was used as the sugar alcohol.

[0045] [ Figure 7 ] Figure 7 Represents 1-OBz in CDCl3 1 H NMR spectrum (600 MHz).

[0046] [ Figure 8 ] Figure 8 This shows the magnified 1-OBz in CDCl3. 1 H NMR spectrum (600 MHz) (6.1-3.3 ppm).

[0047] [ Figure 9] Figure 9 This shows the magnified 1-OBz in CDCl3. 1 H NMR spectrum (600 MHz) (8.5-6.7 ppm).

[0048] [ Figure 10 ] Figure 10 Represents 1-OBz in CDCl3 13 C NMR spectrum (upper) and DEPT 135 spectrum (lower) (150 MHz).

[0049] [ Figure 11 ] Figure 11 Shows the COSY NMR spectrum of 1-OBz in CDCl3.

[0050] [ Figure 12 ] Figure 12 Shows the NOESY NMR spectrum of 1-OBz in CDCl3.

[0051] [ Figure 13 ] Figure 13 Shown is the HSQC NMR spectrum of 1-OBz in CDCl3.

[0052] [ Figure 14 ] Figure 14 Shows the HMBC NMR spectrum of 1-OBz in CDCl3. DETAILED DESCRIPTION

[0053] <<Definition>>

[0054] Throughout this specification, "fructooligosaccharides" (FOS, sometimes referred to as "FOS") refer to oligosaccharides composed of one to three fructose molecules bonded to sucrose. FOS include 1-kestose (sometimes referred to as "GF2" or "tri-ose FOS"), which has one molecule of fructose bonded to a fructose residue in sucrose via a β-2,1 linkage; fructooligosaccharide (sometimes referred to as "GF3" or "tetra-ose FOS"), which has two molecules of fructose bonded to a fructose residue in sucrose via a β-2,1 linkage; and 1-fructofuranosyl-D-kestose (sometimes referred to as "GF4" or "penta-ose FOS"), which has three molecules of fructose bonded to a fructose residue in sucrose via a β-2,1 linkage. FOS are synthesized by β-fructofuranosidase.

[0055] In the present invention, "fructosylated maltitol" is defined as a molecule in which fructose is bonded to maltitol, a sugar alcohol. The number of fructose molecules bonded to maltitol is at least 1 or 2, and the upper limit may be 3 molecules, or more.

[0056] The structure of fructosylated maltitol (sometimes referred to as "F-Mal" in this specification), which is determined in the Examples below and is formed by bonding one molecule of fructose to maltitol, is shown in Formula (I). As shown in Formula (I), the 2-position of fructose is bonded to the 6-position of the glucose moiety of maltitol.

[0057]

[0058] The structure of fructosylated maltitol (sometimes referred to as "FF-Mal" in this specification) in which two fructose molecules are bonded to maltitol is shown in formula (II). As shown in formula (II), it is believed that the 2-position of one molecule of fructose is bonded to the 6-position of the glucose moiety of maltitol, and the 1-position of this fructose is bonded to the 2-position of another molecule of fructose.

[0059]

[0060] In the case of fructosylated maltitol in which three or more fructose molecules are bonded to maltitol, it is believed that the 2-position of another molecule of fructose is further bonded to the 1-position of the fructose at the terminal of the fructosylated maltitol. Taking the example of fructosylated maltitol in which three fructose molecules are bonded to maltitol, it is speculated that the 2-position of another molecule of fructose is bonded to the 1-position of the fructose at the terminal of FF-Mal (the fructose further bonded to the first fructose bonded to maltitol) as shown in formula (III).

[0061]

[0062] <<Method for producing fructosylated maltitol>>

[0063] According to the present invention, a method for producing fructosylated maltitol is provided.

[0064] According to the production method of the present invention, there is provided a method for producing fructosylated maltitol, comprising a step (contact step) of contacting (A) β-fructofuranosidase with sucrose and maltitol, wherein the (A) β-fructofuranosidase has a sequence identity of 60% or more with the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity.

[0065] In the present invention, "β-fructofuranosidase" refers to an enzyme belonging to the GH32 family (glycoside hydrolase family; a group of homologous proteins with high sequence similarity among enzymes that hydrolyze glycosidic bonds of sugars) that has sucrose hydrolysis activity. It should be noted that "β-fructofuranosidase" is sometimes used interchangeably with terms such as "β-fructofuranosidase," "fructosyltransferase," "fructosyl transferase," "sucrase," "β-D-fructofuranosidase," "β-D-fructofuranosidase," "invertase," "invertase," or "invertin," all of which are synonymous.

[0066] The β-fructofuranosidase used in the production method of the present invention may be composed of an amino acid sequence having a sequence identity of 60% or more (preferably 65% ​​or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 98% or more, or 99% or more) to the amino acid sequence of β-fructofuranosidase shown in SEQ ID NO: 1. Here, "identity" refers to, for example, the degree of identity when the sequences to be compared are properly aligned (aligned), and refers to the percentage (%) of correct and identical amino acid occurrences between the sequences. When calculating identity, for example, the presence of gaps in the sequence and the properties of the amino acids can be taken into account (Wilbur, Natl. Acad. Sci. USA 80: 726-730 (1983)). The aforementioned alignment can be performed, for example, using any algorithm. Specifically, publicly available homology search software such as BLAST (Basic local alignment search tool) (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Peasant et al., Methods in Enzymology 183: 63-69 (1990)), and Smith-Waterman (Meth. Enzym., 164, 765 (1988)) can be used. In addition, the calculation of identity can be performed, for example, using the publicly available homology search program described above, for example, by using the default parameters in the homology algorithm BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) of the National Center for Biotechnology Information (NCBI).

[0067] The β-fructofuranosidase used in the production method of the present invention is composed of an amino acid sequence having 60% or more sequence identity with the amino acid sequence of β-fructofuranosidase set forth in SEQ ID NO: 1 and having β-fructofuranosidase activity. In the present invention, "having β-fructofuranosidase activity" can be defined as: the amino acid sequence having about 70% or more, about 80% or more, about 90% or more, about 95% or more, or about 100% or more of the activity of the β-fructofuranosidase consisting of the amino acid sequence set forth in SEQ ID NO: 1 at a temperature of 50° C. to 55° C. and a pH of 5.5. β-fructofuranosidase activity can be evaluated, for example, by mixing a solution containing the protein or polypeptide to be measured with a solution containing sucrose, incubating the mixture, and measuring the amount of glucose generated by the enzyme reaction using high performance liquid chromatography (HPLC), thin layer chromatography (TLC), various glucose quantification kits, or the like. The activity is evaluated based on the value obtained and the amount of enzyme used in the reaction.

[0068] The β-fructofuranosidase used in the production method of the present invention is typically a wild-type enzyme, and may be derived from any organism such as microorganisms, fungi, or plants, preferably from a microorganism belonging to the genus Aspergillus, and more preferably from Aspergillus fijiensis ( Aspergillus fijiensis However, it is also permissible to use β-fructofuranosidase into which a mutation has been artificially introduced in the production method of the present invention.

[0069] Examples of β-fructofuranosidase derived from microorganisms belonging to the genus Aspergillus include those shown in Table 1.

[0070] [Table 1]

[0071] Table 1: Sequences with more than 60% sequence identity to the amino acid sequence of β-fructofuranosidase shown in SEQ ID NO: 1 An example of a β-fructofuranosidase derived from a microorganism (species) belonging to the genus Aspergillus

[0072] Bacteria (source) Accession number Sequence identity (%) Aspergillus aculeatus ANF99482.1 99.69 Aspergillus japonicus 3LF7_A 99.53 Aspergillus japonicum ADK46938.1 99.39 Aspergillus japonicum 3LDK_A 99.37 Aspergillus fijiensis CBS 313.89 XP_040799211.1 99.08 Aspergillus brunneoviolaceus CBS 621.78 XP_025443891.1 98.47 Aspergillus aculeatinus CBS 121060 XP_025508898.1 97.40 Aspergillus japonicum ABD97344.1 97.40 Aspergillus aculeatus ATCC 16872 XP_020051300.1 96.18 Aspergillus uvarum CBS 121591 XP_025487796.1 93.10 Aspergillus indologenus CBS 114.80 PYI32185.1 92.98 Aspergillus japonicus CBS 114.51 XP_025532678.1 91.85 Aspergilus violaceofuscus CBS 115571 PYI19045.1 90.37 Aspergllus homonorphus CBS101889 XP_025551517.1 83.78 Aspergillus saccharolyticus JOP 1030-1 XP_025428139.1 81.69 Aspergillus costaricaensis CBS 115574 XP_025540508.1 64.98 Aspergillus luchuensis IFO 4308 5XH8_A 64.61 Aspergillus elliptticus CBS 707.79 PYH88239.1 64.49 Aspergillus niger AHC54391.1 63.94 Aspergillus costaricaensis CBS 115574 XP_025540508.1 63.94

[0073] The β-fructofuranosidase used in the production method of the present invention may be a substance having substrate specificity for maltitol among sugar alcohols.

[0074] The β-fructofuranosidase used in the production method of the present invention may also have one or more alterations in the amino acid sequence of the β-fructofuranosidase shown in SEQ ID NO: 1 and have β-fructofuranosidase activity. The alterations may be one or more selected from the group consisting of deletion, substitution, insertion, and addition. The alterations may be multiple homologous alterations (e.g., multiple substitutions) or multiple heterologous alterations (e.g., a combination of one or more deletions and one or more substitutions). The number of amino acids to be modified can be, for example, 1-300, 1-275, 1-250, 1-225, 1-200, 1-175, 1-150, 1-125, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1-30, 1-20, or 1-10, preferably 1-60, 1-50, 1-40, 1-30, 1-20, or 1-10, more preferably 1 to several, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1. The number of amino acids to be modified may be the number of mutations produced by a known method such as site-directed mutagenesis or the number of mutations occurring naturally.

[0075] The amino acid changes in the amino acid sequence of β-fructofuranosidase shown in SEQ ID NO: 1 may be conservative changes (e.g., conservative variations). "Conservative changes" or "conservative variations" refer to changes or variations in one or more amino acids that do not substantially alter the function of the protein. The aforementioned amino acid substitutions may also be conservative substitutions. "Conservative substitutions" refer to substitutions in which one or more amino acids are replaced with other amino acids and / or amino acid derivatives that do not substantially alter the function of the protein. In conservative substitutions, the replaced amino acid and the replaced amino acid preferably have similar properties and / or functions. Specifically, it is preferred that the chemical properties such as hydrophobicity and hydrophilicity indicators, polarity, charge, or physical properties such as secondary structure are similar. Amino acids or amino acid derivatives with similar properties and / or functions are well known in the art. For example, non-polar amino acids (hydrophobic amino acids) include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. Amino acids with a positive charge (basic amino acids) include arginine, histidine, lysine, etc., and amino acids with a negative charge (acidic amino acids) include aspartic acid, glutamic acid, etc.

[0076] According to the production method of the present invention, there is provided a method for producing fructosylated maltitol, comprising a step of contacting (B) a polypeptide comprising a portion of the aforementioned β-fructofuranosidase (A) with sucrose and maltitol (contact step), wherein the aforementioned (B) polypeptide comprises an enzymatically active portion having the amino acid sequence of β-fructofuranosidase having the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity.

[0077] The polypeptide used in the production method of the present invention typically contains at least a site (region) having β-fructofuranosidase activity. Examples of sites having β-fructofuranosidase activity in the β-fructofuranosidase used in the production method of the present invention include sites corresponding to positions 20 to 654 from the amino terminus of the amino acid sequence set forth in SEQ ID NO: 1 (region excluding the signal sequence on the N-terminal side), sites corresponding to positions 50 to 558 from the amino terminus of the amino acid sequence set forth in SEQ ID NO: 1 (a characteristic region of enzymes belonging to glucosyl hydrolase family 32), sites corresponding to positions 59 to 428 from the amino terminus of the amino acid sequence set forth in SEQ ID NO: 1 (a characteristic region of the N-terminal side sequence of enzymes belonging to glucosyl hydrolase family 32), sites corresponding to position 60 from the amino terminus of the amino acid sequence set forth in SEQ ID NO: 1 (catalytic center amino acid Asp60), sites corresponding to position 191 from the amino terminus of the amino acid sequence set forth in SEQ ID NO: 1 (catalytic center amino acid Asp191), and sites corresponding to position 292 from the amino terminus of the amino acid sequence set forth in SEQ ID NO: 1 (catalytic center amino acid Glu292). The upper limit of the full length of the polypeptide containing the site (region) having β-fructofuranosidase activity (below or less than) can be, for example, 800 amino acids long, 775 amino acids long, 750 amino acids long, 725 amino acids long, 700 amino acids long, 690 amino acids long, 685 amino acids long, 680 amino acids long, 675 amino acids long, 670 amino acids long, 665 amino acids long, 660 amino acids long, 659 amino acids long, 658 amino acids long, 657 amino acids long, 656 amino acids long. Length, 655 amino acids, 654 amino acids, 653 amino acids, 652 amino acids, 651 amino acids, 650 amino acids, 649 amino acids, 648 amino acids, 647 amino acids, 646 amino acids, 645 amino acids, 644 amino acids, 643 amino acids, 642 amino acids, 641 amino acids, 640 amino acids, 639 amino acids, 638 amino acids, 637 amino acids, 636 amino acids, or 635 amino acids. Furthermore, the total length of the polypeptide used in the production method of the present invention can be arbitrarily adjusted as long as it contains the site having the above-mentioned β-fructofuranosidase activity.

[0078] According to the production method of the present invention, there is provided a method for producing fructosylated maltitol, comprising: a step of contacting a culture of a transformant (C) containing either or both of the aforementioned (A) β-fructofuranosidase and the aforementioned (B) polypeptide with sucrose and maltitol (contact step), wherein the culture of the transformant (C) is obtained by introducing an expression vector containing a polynucleotide encoding the aforementioned (A) β-fructofuranosidase or a polynucleotide encoding the aforementioned (B) polypeptide into a host.

[0079] The culture of the transformant used in the production method of the present invention is obtained by culturing a transformant obtained by introducing a polynucleotide encoding the aforementioned (A) β-fructofuranosidase or the aforementioned (B) polypeptide into a suitable host. The vector introduced into the host may be a vector incorporating the polypeptide. Examples of vectors include phage vectors, plasmid vectors, cosmids, and phagemids, and can be appropriately selected based on the host and operability. Examples of hosts include bacteria such as Escherichia coli and Bacillus subtilis, yeast, molds, and filamentous fungi, and can be appropriately selected based on the type of recombinant vector and operability.

[0080] In the production method of the present invention, the ratio of the amount or concentration of maltitol to sucrose at the start of contact is not particularly limited and can be, for example, 1 or greater. From the viewpoint of increasing the amount of fructosylated maltitol produced, it is preferably 2 or greater, more preferably 3 or greater.

[0081] In the production method of the present invention, the temperature of the contact step is not particularly limited as long as the culture of the (A) β-fructofuranosidase, the (B) polypeptide, or the (C) transformant exhibits enzymatic activity, and can be, for example, 40°C to 60°C, preferably 45°C to 55°C.

[0082] In the production method of the present invention, the time of the contact step can be appropriately set according to the target yield of the fructosylated maltitol to be produced, and can be, for example, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 21 hours or more, 22 hours or more, 23 hours or more, 24 hours or more, 25 hours or more, 26 hours or more, 27 hours or more, 28 hours or more, 29 hours or more, 30 hours or more, 31 hours or more, 32 hours or more, 33 hours or more, 34 hours or more, 35 hours or more, 36 hours or more, 37 hours or more, 38 hours or more, 39 hours or more, 40 hours or more, 41 hours or more, 42 hours or more, 43 hours or more, 44 hours or more, 45 hours or more, 46 hours or more, 47 hours or more, 48 hours or more, 49 hours or more, 50 hours or more, 51 hours or more, 52 hours or more, 53 hours or more, 54 hours or more, 55 hours or more, 56 hours or more, 57 hours or more, 58 hours or more, 59 hours or more, 60 hours or more, 61 hours or more, 62 hours or more, 63 hours or more, 64 hours or more, 65 hours or more, 66 hours or more, 67 hours or more, 68 hours or more, 69 hours or more, 7 The time period may be 7 hours or more, 48 hours or more, 49 hours or more, 50 hours or more, 51 hours or more, 52 hours or more, 53 hours or more, 54 hours or more, 55 hours or more, 60 hours or more, 65 hours or more, 70 hours or more, 75 hours or more, 80 hours or more, 85 hours or more, 90 hours or more, 95 hours or more, or 100 hours or more. It may also be any time period of 2 hours or more and 100 hours or less, 10 hours or more and 100 hours or less, 20 hours or more and 100 hours or less, 30 hours or more and 100 hours or less, 40 hours or more and 100 hours or less, or 50 hours or more and 100 hours or less. From the viewpoint of improving the production efficiency of fructosylated maltitol, it is preferably 55 hours or more and 100 hours or less.

[0083] The production method of the present invention may include a step of concentrating the sugar solution containing fructosylated maltitol (concentration step) after the contact step. The concentration step can be carried out according to a conventional method.

[0084] The production method of the present invention may include a step of purifying the fructosylated maltitol in the sugar solution or the concentrated solution (purification step) after the contact step or the concentration step. The purification step can be carried out according to a conventional method.

[0085] <<Enzyme>>

[0086] According to the present invention, there is also provided an enzyme agent for producing fructosylated maltitol, comprising the aforementioned (A) β-fructofuranosidase, the aforementioned (B) polypeptide, or a culture of the aforementioned (C) transformant. The enzyme agent of the present invention can be implemented according to the description of the production method of the present invention.

[0087] <<Fructosylated maltitol>>

[0088] According to the present invention, there is also provided a fructosylated maltitol characterized in that it is maltitol to which one, two or three molecules of fructose are bonded.

[0089] The fructosylated maltitol of the present invention is represented by the aforementioned formula (I) when one molecule of fructose is bonded to maltitol, and is a product in which the 2-position of fructose is bonded to the 6-position of the glucose moiety of maltitol.

[0090] In the case where two molecules of fructose are bonded to maltitol, the fructosylated maltitol of the present invention is represented by the aforementioned formula (II), and it is considered that the 2-position of one molecule of fructose is bonded to the 6-position of the glucose moiety of maltitol, and the 1-position of this fructose is bonded to the 2-position of another molecule of fructose.

[0091] When three fructose molecules are bonded to maltitol in the fructosylated maltitol of the present invention, as shown in the above formula (III), it is considered that the 2-position of another molecule of fructose is bonded to the 1-position of the fructose at the terminal of FF-Mal.

[0092] The fructosylated maltitol of the present invention can be produced according to the description of the production method of the present invention.

[0093] <<Enzyme reaction composition containing fructosylated maltitol>>

[0094] According to the present invention, there is also provided an enzyme reaction composition comprising β-fructofuranosidase as an enzyme and sucrose and maltitol as substrates.

[0095] According to the enzyme reaction composition of the present invention, an enzyme reaction composition is provided, which comprises (D) β-fructofuranosidase as an enzyme, and sucrose and maltitol as substrates, wherein the ratio of the aforementioned fructosylated maltitol to the total sugars in the composition is 5% by mass or more, and the aforementioned (D) β-fructofuranosidase has a sequence identity of 60% or more with the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity.

[0096] In addition, according to the enzyme reaction composition of the present invention, there is provided an enzyme reaction composition comprising: (E) a polypeptide comprising a portion of the aforementioned (D) β-fructofuranosidase as an enzyme, and sucrose and maltitol as substrates, wherein the ratio of the aforementioned fructosylated maltitol to the total sugars in the composition is 5% by mass or more, and the aforementioned (E) polypeptide comprises an enzymatically active portion having the amino acid sequence of β-fructofuranosidase having the amino acid sequence shown in SEQ ID NO: 1, and has β-fructofuranosidase activity.

[0097] According to the enzyme reaction composition of the present invention, there is also provided an enzyme reaction composition comprising: a culture of a transformant (F) comprising either or both of the (D) β-fructofuranosidase and the (E) polypeptide as an enzyme; and sucrose and maltitol as substrates, wherein the ratio of the fructosylated maltitol in the composition to the total sugars is 5% by mass or more, and the culture of the transformant (F) is obtained by introducing an expression vector comprising a polynucleotide encoding the (D) β-fructofuranosidase or a polynucleotide encoding the (E) polypeptide into a host.

[0098] In the enzyme reaction composition of the present invention, the ratio of the fructosylated maltitol to the total sugars in the composition is 5% by mass or more, and can be further 6% by mass or more, 7% by mass or more, 8% by mass or more, 9% by mass or more, or 10% by mass or more.

[0099] In addition to the above, the enzyme reaction composition of the present invention can also be produced according to the description of the production method of the present invention.

[0100] <<Method for fructosylation of maltitol>>

[0101] According to the present invention, a method for fructosylation of maltitol is also provided.

[0102] According to the method for fructosylation of the present invention, a method for fructosylation of maltitol is provided, which includes a step of contacting (G) β-fructofuranosidase with sucrose and maltitol (contact step), wherein the aforementioned (G) β-fructofuranosidase has a sequence identity of 60% or more with the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity.

[0103] According to the method for fructosylation of the present invention, there is also provided a method for fructosylation of maltitol, comprising a step of contacting (H) a polypeptide comprising a portion of the aforementioned (G) β-fructofuranosidase with sucrose and maltitol (contact step), wherein the aforementioned (H) polypeptide comprises an enzymatically active portion having the amino acid sequence of β-fructofuranosidase having the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity.

[0104] According to the method for fructosylation of the present invention, there is also provided a method for fructosylation of maltitol, comprising the step of contacting a culture of (I) a transformant containing either or both of the (G) β-fructofuranosidase and the (H) polypeptide with sucrose and maltitol (contact step), wherein the culture of the transformant (I) is a culture of a transformant obtained by introducing an expression vector containing a polynucleotide encoding the (G) β-fructofuranosidase or a polynucleotide encoding the (H) polypeptide into a host.

[0105] In addition to the above, the fructosylation method of the present invention can also be carried out according to the description of the production method of the present invention.

[0106] <<β-Fructofuranosidase>>

[0107] The present invention also provides a use of a β-fructofuranosidase for producing fructosylated maltitol, wherein the β-fructofuranosidase has a sequence identity of 60% or greater with the amino acid sequence set forth in SEQ ID NO: 1 and exhibits β-fructofuranosidase activity. In addition to the above, the use of the present invention can also be implemented as described in the production method of the present invention.

[0108] In addition to the above, the fructosylation method of the present invention can also be carried out according to the description of the production method of the present invention.

[0109] Example

[0110] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

[0111] Example 1: Study on the fructosylation of sugar alcohols (1)

[0112] In Example 1, the fructosylation of various sugar alcohols was studied using β-fructofuranosidase.

[0113] (1) Method

[0114] The Aspergillus Fijiensis-derived bacteria ( Aspergillus fijiensis ATCC20611) was used to investigate the fructosylation of each sugar alcohol listed in Table 3 under the reaction conditions shown in Table 4. The reaction solutions were analyzed for the composition of each sugar using high performance liquid chromatography (HPLC) under the analysis conditions shown in Table 5. The results of the composition analysis were calculated based on the HPLC peak area to determine the ratio (% (w / w)) of each sugar relative to the total sugars.

[0115] [Table 2]

[0116] Table 2: Amino acid sequence of AfBFFase (SEQ ID NO: 1)

[0117]

[0118] *Amino acids 1 to 19 (underlined in the sequence) represent the signal sequence.

[0119] [Table 3]

[0120] Table 3: Sugar alcohols

[0121]

[0122] [Table 4]

[0123] Table 4: Reaction conditions

[0124] AfBFFase 1.0 μg sucrose 1.0M Sugar alcohols* 1.0M solvent 300 μL (50 mM sodium phosphate buffer, pH 5.5) Reaction temperature 50％ Reaction time 48 hours

[0125] *Any of the sugar alcohols listed in Table 3

[0126] [Table 5]

[0127] Table 5: Analysis conditions

[0128]

[0129] (2) Results

[0130] The results are shown in Tables 6 to 9 and Figures 1 to 4 When maltitol was used as the disaccharide alcohol, maltitol (F-Mal) to which one molecule of fructose was bonded and maltitol (F-Mal) to which one molecule of fructose was bonded were produced as fructosylated maltitol (Table 6, Figure 1 On the other hand, when lactitol and isomalt were used as disaccharide alcohols, no fructosylated lactitol and fructosylated isomalt were produced (Table 7, Table 8, Figure 2 、 Figure 3 ). In addition, when xylitol was used as the monosaccharide alcohol, no fructosylated xylitol was produced (Table 9, Figure 4 ). Similarly, for monosaccharide alcohols other than xylitol, no fructosylated sugar alcohols were produced (data not shown). These results indicate that AfBFFase performs substrate-specific fructosylation on maltitol to produce fructosylated maltitol. It should be noted that, although not subject to the following theoretical constraints, AfBFFase shows reactivity in the case of maltitol, but does not show reactivity in the case of isomalt, which also has a closed-loop sugar. Therefore, it is speculated that the reducing sugar part of maltitol is involved in the contact of AfBFFase. In addition, lactitol is different from sucrose and maltitol, and the closed-loop sugar part is galactose. Therefore, it is speculated that glucose is specifically involved in the contact of AfBFFase with maltitol.

[0131] [Table 6]

[0132] Table 6: Ratio of each sugar relative to total sugars in the reaction solution when maltitol was used (% (w / w))

[0133]

[0134] (Abbreviation) Gul: glucose, Suc: sucrose, Mal: ​​maltitol, F0S: oligofructose,

[0135] F-Mal: maltitol to which one molecule of fructose is bonded (fructosylated maltitol),

[0136] FF-Mal: Maltitol with two molecules of fructose bonded to it (fructosylated maltitol),

[0137] -: Unable to detect (HPLC peak area is too small to be calculated)

[0138] [Table 7]

[0139] Table 7: Ratio of each sugar relative to total sugars in the reaction solution when lactitol was used (% (w / w))

[0140]

[0141] (Abbreviation) Gul: glucose, Suc: sucrose, Lac: lactitol, FOS: oligofructose,

[0142] F-Lac: Lactitol to which one molecule of fructose is bonded (fructosylated lactitol),

[0143] FF-Lac: Lactitol with two fructose molecules bonded to it (fructosylated lactitol)

[0144] -: Unable to detect (HPLC peak area is too small to be calculated)

[0145] [Table 8]

[0146] Table 8: Ratio of each sugar relative to total sugars in the reaction solution when isomalt was used (% (w / w))

[0147]

[0148] (Abbreviation) Gul: glucose, Suc: sucrose, Pal: isomalt, FOS: oligofructose,

[0149] F-Pal: Isomalt to which one molecule of fructose is bonded (fructosylated isomalt),

[0150] FF-Pal: Isomalt to which two molecules of fructose are bonded (fructosylated isomalt)

[0151] -: Unable to detect (HPLC peak area is too small to be calculated)

[0152] [Table 9]

[0153] Table 9: Ratio of each sugar relative to total sugars in the reaction solution when xylitol was used (% (w / w))

[0154]

[0155] (Abbreviation) Gul: glucose, Xyl: xylitol, Suc: sucrose FOS: oligofructose,

[0156] F-Xyl: xylitol to which one molecule of fructose is bonded (fructosylated xylitol),

[0157] FF-Xyl: Xylitol with two fructose molecules bonded to it (fructosylated xylitol)

[0158] -: Unable to detect (HPLC peak area is too small to be calculated)

[0159] Example 2: Study on the fructosylation of sugar alcohols (2)

[0160] In Example 2, in the fructosylation of maltitol by β-fructofuranosidase, the effect of the amount of maltitol as a substrate on the amount of fructosylated maltitol produced was examined.

[0161] (1) Method

[0162] The Aspergillus Fijiensis-derived bacteria ( Aspergillus fijiensis ATCC20611) (SEQ ID NO: 1), and the effect of the reaction conditions of various maltitol concentrations shown in Table 10 on the production of fructosylated maltitol was examined. Each reaction solution was analyzed for the composition of each sugar using high-performance liquid chromatography (HPLC) under the analysis conditions shown in Table 11. The results of the composition analysis were used to calculate the ratio (% (w / w)) of each sugar relative to the total sugars and the ratio (% (w / w)) of each FOS relative to the total FOS based on the HPLC peak area.

[0163] [Table 10]

[0164] Table 10: Reaction conditions

[0165] AfBFFase 1.0 μg Sucrose: Maltitol* 1:1 (1.0M:1.0M), 1:2 (0.67M:1.33M) or 1:3 (0.5M:1.5M) solvent 300 μL (50 mM sodium phosphate buffer, pH 5.5) Reaction temperature 50℃ Reaction time 48 hours

[0166] *The ratio of sucrose to maltitol was adjusted so that the total concentration of the sucrose and maltitol was 2M.

[0167] [Table 11]

[0168] Table 11: Analysis conditions

[0169]

[0170] (2) Results

[0171] The results are shown in Tables 12, 13 and Figure 5It was confirmed that when the concentration of maltitol was increased (the ratio of maltitol to sucrose was 1:1, 1:2, and 1:3), the production amount of maltitol (F-Mal), which is fructosylated maltitol and to which one molecule of fructose is bound, increased in a concentration-dependent manner (Table 12, Table 13, Figure 5 ). These results indicate that the fructosylation of maltitol by AfBFFase is concentration-dependent. It should be noted that in Table 12, the ratio of maltitol (FF-Mal) to which two molecules of fructose are bonded relative to the total sugar is reduced when the ratio of maltitol to sucrose is 1:3 compared to the ratios of 1:1 and 1:2, but this is presumably due to the depletion of sucrose, which serves as a source of fructose. On the other hand, in Table 13, the ratio of FF-Mal to total FOS increases as the ratio of maltitol to sucrose increases to 1:1, 1:2, and 1:3. It is presumed that this is because, when sucrose is depleted, fructose is supplied by tri-sugar FOS and tetra-sugar FOS, which function as a donor, whereas FF-Mal does not serve as a fructose donor, and therefore its ratio relative to total FOS increases.

[0172] [Table 12]

[0173] Table 12: Ratio of each sugar to total sugar when using maltitol at various concentrations (% (w / w))

[0174]

[0175] (Abbreviation) Gul: glucose, Suc: sucrose, Mal: ​​maltitol, FOS: oligofructose,

[0176] F-Mal: maltitol to which one molecule of fructose is bonded (fructosylated maltitol),

[0177] FF-Mal: Isomalt to which two molecules of fructose are bonded (fructosylated maltitol)

[0178] -: Unable to detect (HPLC peak area is too small to be calculated)

[0179] [Table 13]

[0180] Table 13: Ratio of each FOS relative to total FOS when using maltitol at various concentrations (% (w / w))

[0181]

[0182] (Abbreviation) Gul: glucose, Suc: sucrose, Mal: ​​maltitol, FOS: oligofructose,

[0183] F-Mal: maltitol to which one molecule of fructose is bonded (fructosylated maltitol),

[0184] FF-Mal: Isomalt to which two molecules of fructose are bonded (fructosylated maltitol)

[0185] -: Unable to calculate (HPLC peak area is too small to be calculated)

[0186] Example 3: Study on the fructosylation of sugar alcohols (3)

[0187] Example 3 shows a production example of fructosylation of maltitol using β-fructofuranosidase.

[0188] (1) Method

[0189] The Aspergillus Fijiensis-derived bacteria ( Aspergillus fijiensis ATCC20611) (SEQ ID NO: 1), and fructosylated maltitol was produced by the following steps (i) to (iii).

[0190] (i) The reaction composition liquid shown in Table 14 was prepared.

[0191] (ii) 150 μL of the above composition solution was taken and tested under the reaction conditions shown in Table 15.

[0192] (iii) The reaction solution at each reaction time was diluted 10-fold with distilled water and analyzed for components using RI-HPLC. Table 16 shows the analysis conditions.

[0193] [Table 14]

[0194] Table 14: Reaction composition

[0195] AfBFFase 20 mg (14.7 million IU / g) sucrose 133g Maltitol 229g solvent 500mL (50mM sodium phosphate buffer, pH 5.5)

[0196] [Table 15]

[0197] Table 15: Reaction conditions

[0198]

[0199] [Table 16]

[0200] Table 16: Analysis conditions

[0201]

[0202] (2) Results

[0203] The results are shown in Table 17 and Figure 6As shown, the ratio of fructosylated maltitol to total sugars increased with the reaction time, and the total amount of maltitol bound to one molecule of fructose (F-Mal) and maltitol bound to two molecules of fructose (FF-Mal) reached a production amount exceeding 10% after 54 hours.

[0204] [Table 17]

[0205] Table 17: Ratio of total sugar at each reaction time (% (w / w))

[0206]

[0207] Table 17: Ratio of total sugars at each reaction time (% (w / w)) (Continued)

[0208]

[0209] Example 4: Study on the fructosylation of sugar alcohols (4)

[0210] Example 4 shows a production example of fructosylation of maltitol using β-fructofuranosidase.

[0211] The bacteria derived from Aspergillus Fiji ( Aspergillus fijiensis ATCC20611) (SEQ ID NO: 1), and fructosylated maltitol was produced by the following steps (i) to (iii).

[0212] (i) The reaction composition liquid shown in Table 18 was prepared.

[0213] (ii) 150 μL of the above composition solution was taken and tested under the reaction conditions shown in Table 19.

[0214] (iii) At each reaction time, the reaction solution was diluted 10-fold with distilled water and analyzed for components using RI-HPLC. Table 20 shows the analysis conditions.

[0215] [Table 18]

[0216] Table 18: Reaction composition

[0217] AfBFFase 80mg sucrose 452g Maltitol 916g solvent 2000mL (50mM sodium phosphate buffer, pH 5.5)

[0218] [Table 19]

[0219] Table 19: Reaction conditions

[0220]

[0221] [Table 20]

[0222] Table 20: Analysis conditions

[0223]

[0224] Example 5: Study on the fructosylation of sugar alcohols (5)

[0225] In Example 5, the structure of maltitol (F-Mal) to which one molecule of fructose is bonded was determined by NMR analysis.

[0226] A mixture containing F-Mal (120 mg, 0.237 mmol) was dissolved in dichloromethane (10 ml). The solution was cooled to 0°C, benzoyl chloride (551 μL, 4.74 mmol), triethylamine (991 μL, 7.11 mmol) and 4-dimethylaminopyridine (145 mg, 1.19 mmol) were added, and the mixture was stirred at room temperature for 24 hours to react. 1 M hydrochloric acid was then added to terminate the reaction, ethyl acetate was added, and the organic layer was washed with water and saturated saline solution in sequence, and then dried over sodium sulfate. After filtering and separating the desiccant, the solvent was removed by distillation under reduced pressure. The residue was then subjected to column chromatography (silica gel, hexane / ethyl acetate = 2 / 1 to 1 / 1), followed by gel filtration chromatography (chloroform, 3.5 ml / min) to obtain a mixture containing F-Mal. (-OBz) The yellow amorphous substance was purified by high performance liquid chromatography (silica gel, hexane / ethyl acetate = 4 / 3, 3.5 ml / min) to obtain F-Mal as a white amorphous substance. (-OBz) (154 mg, 37% yield). Then, F-Mal (-OBz) NMR analysis (Bruker Avance III600, Bruker) yielded 1 HNMR spectroscopy, 13 CNMR spectrum, COSY NMR spectrum, NOESY NMR spectrum, HSQC NMR spectrum and HMBC NMR spectrum. In addition, the peaks of each NMR spectrum are shown in Figures 7 to 14 .

[0227] 1H NMR(600MHz,CDCl3):δ8.20-8.18(m,2H),8.06-8.04(m,2H),7.93-7.85(m,10H),7.84-7.82(m,2H),7.80-7.77(m,4H),7.73-7.69(m,4H),7.60-7.57(m,1H),7.50-7.29(m,20H),7.25-7.17(m,15H),7.11-7.08(m,2H),6.09-6.07(m,4H,G3+S2+S5+F3),6.02(dd,J=6.0,5.4Hz,1H,S3),5.85(dd,J=6.6,6.0Hz,1H,F4),5.83(d,J=3.6Hz,1H,G1),5.79(dd,J=10.2,9.6Hz,1H,G4),5.49(dd,J=10.2,3.6Hz,1H,G2),5.05(dd,J=12.0,8.4Hz,1H,S1),4.96(dd,J=6.0,3.0Hz,1H,S4),4.88(dd,J=12.0,3.6Hz,1H,S1),4.76(dd,J=12.0,3.0Hz,1H,S6),4.72-4.68(m,3H,S6+F6),4.63(m,1H,G5),4.54-4.51(m,1H,F5),4.33(d,J=12.0Hz,1H,F1),4.31(d,J=12.0Hz,1H,F1),3.69(dd,J=9.0,1.5Hz,1H,G6),3.39(dd,J=9.0,1.5Hz,1H,G6). 13C NMR (150MHz, CDCl3): δ166.0, 165.9, 165.8, 165.7, 165.54, 165.53, 165. 49, 165.4, 165.3, 165.1, 165.0, 164.2, 133.5, 133.4, 133.2, 133.1, 133.0 3, 133.00, 132.91, 132.86, 132.84, 132.82, 132.80, 130.4, 130.1, 129.9 3, 129.90, 129.72, 129.70, 129.69, 129.67, 129.45, 129.43, 129.34, 129. HRMS (MALDI-TOF): m / z calculated based on C 102 H 82 NaO 28 [M+Na] + : 1777.49, measured value: 1777.92.

[0228] The NMR spectra were analyzed and confirmed to be the F-Mal structure represented by formula (IV). Specifically, it is a structure in which the 6-position (G6) of the glucose moiety of maltitol is bonded to the 2-position (F2) of fructose. G6 exhibits an upfield shift, and the ppm values ​​of the two geminal H atoms of G6 are very far apart, so there is no perceived inconsistency (since G6 is sandwiched between the glucose and fructose rings, the steric effect is significant).

[0229]

[0230] Industrial applicability

[0231] According to the present invention, fructosylated maltitol can be produced simply and efficiently. According to the present invention, fructosylated maltitol having a sweet taste and a food containing the same can also be provided.

Claims

1. A method for producing fructosylated maltitol, comprising a step of contacting a culture of (A) β-fructofuranosidase, (B) a polypeptide comprising a portion thereof, or (C) a transformant comprising either or both thereof with sucrose and maltitol, i.e., a contacting step. The (A) β-fructofuranosidase has a sequence identity of more than 60% with the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity. The polypeptide (B) comprises an enzymatically active portion of the amino acid sequence of β-fructofuranosidase having the amino acid sequence shown in SEQ ID NO: 1, and has β-fructofuranosidase activity. The culture of the transformant (C) is obtained by introducing an expression vector containing a polynucleotide encoding the (A) β-fructofuranosidase or a polynucleotide encoding the (B) polypeptide into a host.

2. The manufacturing method according to claim 1, wherein β-Fructofuranosidase has substrate specificity for maltitol among sugar alcohols.

3. The manufacturing method according to claim 1 or 2, wherein: The ratio of the amount or concentration of maltitol to sucrose at the start of contact is 1 or more.

4. The manufacturing method according to claim 1 or 2, wherein: The temperature of the contact process is 40°C to 60°C.

5. The manufacturing method according to claim 1 or 2, wherein: The contact step takes 8 hours or longer.

6. The manufacturing method according to claim 1 or 2, wherein: The host is selected from the group consisting of bacteria, yeast, mold and filamentous fungi.

7. The manufacturing method according to claim 1 or 2, wherein: β-Fructofuranosidase is derived from a microorganism of the genus Aspergillus.

8. The manufacturing method according to claim 1 or 2, wherein: β-Fructofuranosidase is derived from Aspergillus fijiensis.

9. An enzyme agent for producing fructosylated maltitol, comprising: a culture of a β-fructofuranosidase or a polypeptide comprising a portion thereof, or a transformant comprising either or both thereof; The β-fructofuranosidase has a sequence identity of more than 60% with the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity. The polypeptide comprises an enzymatically active portion of the amino acid sequence of β-fructofuranosidase having the amino acid sequence shown in SEQ ID NO: 1, and has β-fructofuranosidase activity. The culture of the transformant is a culture of the transformant obtained by introducing an expression vector containing a polynucleotide encoding the β-fructofuranosidase or a polynucleotide encoding the polypeptide into a host.

10. Fructosylated maltitol, which is maltitol to which one or two molecules of fructose are bonded.

11. The fructosylated maltitol according to claim 10, wherein Maltitol bonded with one molecule of fructose is represented by formula (I):

12. The fructosylated maltitol according to claim 10, wherein Maltitol bonded with two molecules of fructose is represented by formula (II), 13. An enzyme reaction composition comprising: (D) β-fructofuranosidase as an enzyme, (E) a polypeptide comprising a portion thereof, or (F) a culture of a transformant comprising either or both thereof, and sucrose and maltitol as substrates, wherein the proportion of fructosylated maltitol relative to the total sugars in the composition is 5% or more, and The (D) β-fructofuranosidase has a sequence identity of more than 60% with the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity. The polypeptide (E) comprises an enzymatically active portion of the amino acid sequence of β-fructofuranosidase having the amino acid sequence shown in SEQ ID NO: 1, and has β-fructofuranosidase activity. The culture of the (F) transformant is obtained by introducing an expression vector containing a polynucleotide encoding the (D) β-fructofuranosidase or a polynucleotide encoding the (E) polypeptide into a host.

14. Use of β-fructofuranosidase for producing fructosylated maltitol, wherein: The β-fructofuranosidase has a sequence identity of more than 60% with the amino acid sequence shown in SEQ ID NO: 1 and has β-fructofuranosidase activity.

Images