Method for producing oligosaccharide and oligosaccharide production system

Abstract

To provide a new method for producing oligosaccharide and the like.SOLUTION: A method for producing an oligosaccharide comprises: selecting an oligosaccharide to be produced from among a plurality of oligosaccharides; selecting one or more carbon sources from among a plurality of carbon sources according to the type of the selected oligosaccharide; and producing the selected oligosaccharide by contacting the selected carbon source with at least one selected from the group consisting of Zygosaccharomyces yeast, a glycosyltransferase derived from Zygosaccharomyces yeast, and a transformant transformed so as to express the glycosyltransferase.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method for producing oligosaccharides and a system for producing oligosaccharides. [Background technology]

[0002] Oligosaccharides are compounds with a structure in which multiple sugars are linked together and have effects such as intestinal regulation. Many oligosaccharides are produced using the transfer reaction of hydrolases produced by microorganisms. For example, isomaltooligosaccharides are produced from maltose, galactooligosaccharides from lactose, and fructooligosaccharides from sucrose.

[0003] To date, molds and yeasts capable of producing oligosaccharides have been reported. Yeasts generally grow faster than molds, have uniform cell morphology, and are easy to cultivate, so the industrial value of finding yeasts that contribute to oligosaccharide production is high. Non-Patent Documents 1 and 2 report that Zararia ( Zalaria ) yeasts are disclosed. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Yoshikawa et al. J Appl Microbiol, 132(2), 1104-1111 (2022) [Non-patent document 2] Yoshikawa et al. BMC Genomic DATA, 23(78), 1-7 (2022) Summary of the Invention [Problem to be solved by the invention]

[0005] Although the above-mentioned Non-Patent Documents 1 and 2 disclose that yeasts of the genus Zararia produce fructooligosaccharides, the methods for producing oligosaccharides using yeasts are still limited.

[0006] An object of the present invention is to provide a novel method for producing oligosaccharides. [Means for solving the problem]

[0007] As a result of extensive research into oligosaccharide-producing yeast, the present inventors have found that yeasts belonging to the genus Zalaria can produce a variety of different oligosaccharides from a variety of different carbon sources. This has led to the discovery that a completely different method and system for producing oligosaccharides can be provided, and has led to the completion of the present invention.

[0008] That is, the present invention includes the following embodiments. [1] A method for producing oligosaccharides, comprising: selecting an oligosaccharide to be produced from a plurality of types of oligosaccharides; selecting one or more carbon sources from a plurality of carbon sources according to the type of oligosaccharide selected; producing the selected oligosaccharide by contacting the selected carbon source with at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express the glycosyltransferase; A method comprising: [2] The oligosaccharides produced are selected from fructooligosaccharides, isomaltooligosaccharides, and gentiooligosaccharides. [1] The method described in [1]. [3] the one or more carbon sources are selected from monosaccharides or disaccharides; The method according to [1] or [2]. [4] The selected carbon source comprises at least one selected from sucrose, maltose, and cellobiose; The method according to any one of [1] to [3]. [5] When the selected oligosaccharides include fructooligosaccharides, at least sucrose is selected as the carbon source; When the selected oligosaccharides include isomaltooligosaccharides, at least maltose is selected as the carbon source; When the selected oligosaccharides include gentiooligosaccharides, at least cellobiose is selected as the carbon source. The method according to any one of [1] to [4]. [6] The yeast comprises at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express the glycosyltransferase, one or more carbon sources selected from a plurality of types of oligosaccharides according to the type of oligosaccharide selected are added to produce the type of oligosaccharide corresponding to the added carbon source; An oligosaccharide production system capable of producing two or more types of oligosaccharides. [7] A method for producing isomaltooligosaccharides, comprising contacting maltose with at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express the glycosyltransferase, thereby producing isomaltooligosaccharides. [8] The yeast comprises at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express the glycosyltransferase, Producing isomaltooligosaccharides from maltose Isomaltooligosaccharide production system. [9] A method for producing oligosaccharides, comprising contacting cellobiose with at least one selected from the group consisting of a Zararia yeast, a glycosyltransferase derived from a Zararia yeast, and a transformant transformed to express the glycosyltransferase, thereby producing an oligosaccharide containing gentiobiose, such as a gentiooligosaccharide.

[10] The yeast comprises at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express the glycosyltransferase, Producing oligosaccharides containing gentiobiose, such as gentiooligosaccharides, from cellobiose; Oligosaccharide production system. [Effects of the Invention]

[0009] According to the present invention, a novel method for producing oligosaccharides can be provided. [Brief explanation of the drawings]

[0010]

Figure 1

Figure 2

[0011] Below, we will explain in detail the form for implementing the present invention (hereinafter referred to as the ``present embodiment''), but the present invention is not limited to this and various modifications are possible within the scope of the gist of the present invention.

[0012] [Method of producing oligosaccharides] One aspect of this embodiment is a method for producing oligosaccharides, comprising: selecting an oligosaccharide to be produced from a plurality of types of oligosaccharides; selecting one or more carbon sources from a plurality of types of carbon sources depending on the type of selected oligosaccharide; and producing the selected oligosaccharide by contacting the selected carbon source with at least one selected from the group consisting of a Zararia yeast, a glycosyltransferase derived from a Zararia yeast (hereinafter sometimes simply referred to as a "glycosyltransferase" in this specification), and a transformant transformed to express the glycosyltransferase.

[0013] As shown in the Examples below, Salarria yeast can utilize multiple carbon sources, such as maltose, sucrose, and cellobiose, and can produce different oligosaccharides depending on the type of carbon source utilized. Therefore, the oligosaccharide production method of this embodiment (hereinafter also referred to as the "method of this embodiment") is completely different from conventional oligosaccharide production methods in which a specific mold or yeast is selected depending on the type of oligosaccharide to be produced. By using at least one selected from the group consisting of Salarria yeast, glycosyltransferases derived from Salarria yeast, and transformants transformed to express glycosyltransferases, a variety of oligosaccharides can be produced from a variety of carbon sources. From this perspective, the method of this embodiment can also be considered a multi-production method for oligosaccharides.

[0014] (Zalaria yeast) The genus Zararia yeast is a black yeast that was first reported in 2017. The genus Zararia yeast includes, but is not limited to: Zalaria obscura , Zalaria alba , and Zalaria Among these, the yeasts of the genus Zararia include: Zalaria sp. Him3 is preferred. Zalaria Details of sp. Him3 are described in Non-Patent Documents 1 and 2.

[0015] In the method of this embodiment, a glycosyltransferase derived from a yeast belonging to the genus Zararia can also be used. The glycosyltransferase used is preferably β-fructofuranosidase. The glycosyltransferase derived from a yeast of the genus Zararia is preferably a β-fructofuranosidase derived from a yeast of the genus Zararia, and more preferably Zalaria It is a β-fructofuranosidase derived from sp. Him3.

[0016] Glycosyltransferases derived from Zararia yeast can be obtained by various known methods. For example, extracellular glycosyltransferases can be obtained from the culture medium of Zararia yeast, cell surface glycosyltransferases can be obtained from the cell surface of Zararia yeast, or intracellular glycosyltransferases can be obtained by disrupting Zararia yeast. Alternatively, glycosyltransferases can be obtained from a transformant that has been transformed to express a glycosyltransferase derived from Zararia yeast, as described below. Glycosyltransferases derived from Zararia yeast may be glycosyltransferases produced by natural Zararia yeast, or may be glycosyltransferases obtained by mutating the natural enzyme. The mutations made to the glycosyltransferase may be additions, deletions, and / or substitutions of one or more amino acids, and "multiple" may be 2 or less, 5 or less, 8 or less, 10 or less, 15 or less, or 20 or less.

[0017] The purification conditions and degree of purification of glycosyltransferases derived from Zararia yeast are not particularly limited, and common purification techniques can be used. After culturing Zararia yeast or a transformant transformed to express a glycosyltransferase, the cells are separated by separation means such as centrifugation or membrane treatment. If the glycosyltransferase is contained in the culture supernatant, it can be collected and used as a crude enzyme solution. Furthermore, if the glycosyltransferase is expressed in large amounts on the cell surface, the cell suspension can be used as a crude enzyme solution. If the glycosyltransferase is contained intracellularly, the cells can be physically disrupted using a homogenizer or ultrasonic treatment, or enzymatically treated with a cell wall-lytic enzyme or the like to obtain an intracellular extract, which can then be used as a crude enzyme solution. These crude enzyme solutions can also be purified to a high degree by appropriately combining ammonium sulfate salting-out, dialysis, gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, adsorption chromatography, affinity chromatography, and the like.

[0018] In the method of this embodiment, the glycosyltransferase derived from a yeast of the genus Zararia preferably comprises at least one selected from the group consisting of extracellular glycosyltransferases obtained from a culture medium of a yeast of the genus Zararia and cell surface glycosyltransferases obtained from the cell surface of a yeast of the genus Zararia; ZalariaExtracellular glycosyltransferases obtained from the culture medium of sp. Him3, and Zalaria It is more preferable that the glycosyltransferase comprises at least one selected from the group consisting of cell surface glycosyltransferases isolated from the cell surface of sp. Him3.

[0019] When a cell surface glycosyltransferase is used as the glycosyltransferase, a cell suspension of a Zalaria yeast can be used as an enzyme solution containing the cell surface glycosyltransferase. In this embodiment, the cell suspension can be used as an enzyme solution without purifying the glycosyltransferase, allowing for easy preparation of the glycosyltransferase.

[0020] In the method of this embodiment, a transformant transformed to express the above-described glycosyltransferase can also be used. The transformant can be obtained by transforming a host cell with a recombinant plasmid obtained by incorporating DNA encoding the above-described glycosyltransferase into a vector. Recombinant DNA technology is well known in the art.

[0021] The host cell may be any cell that can stably and autonomously replicate the recombinant DNA and can express the traits of the foreign DNA. The host cell is preferably a microbial cell. The microbial cell may be a eukaryotic (e.g., yeast) cell or a prokaryotic cell. An example of a host cell is Escherichia coli ( Escherichia coli ), but is not limited to E. coli, Escherichia bacteria, Bacillus subtilis ( Bacillus subtilis Examples of suitable bacteria include bacteria of the genus Bacillus, such as Bacillus sp., bacteria of the genus Pseudomonas, bacteria of the genus Corynebacterium, bacteria of the genus Streptomyces, yeasts of the genus Saccharomyces, Pichia, Candida, and the like, and filamentous fungi of the genus Aspergillus.

[0022] In the method of this embodiment, at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express a glycosyltransferase can be used, and these can be used alone or in combination of two or more. In the method of this embodiment, it is preferable to use at least one selected from the group consisting of a yeast of the genus Zararia and a glycosyltransferase derived from a yeast of the genus Zararia.

[0023] The form of at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant is not particularly limited, and examples include a culture solution containing at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant, a bacterial cell concentrate and / or pellet obtained by concentrating the culture solution by centrifugation, membrane treatment, etc., dried bacterial cells, disrupted bacterial cells, a crude enzyme solution, a purified enzyme solution, an enzyme powder, etc. These may be prepared according to known methods.

[0024] For example, when using a yeast of the genus Zararia, the yeast of the genus Zararia is cultured, and the resulting culture solution may be used as is, or may be subjected to treatment such as centrifugation, membrane treatment, drying, or disruption as necessary, and used as a concentrated cell solution or pellet, dried cells, a cell disruption solution, etc. The cells may be used as live cells, or may be killed by treatment with an organic solvent, freeze-drying, etc.

[0025] (Selection of oligosaccharides) The method of this embodiment includes selecting an oligosaccharide to be produced from a selection of multiple types of oligosaccharides. As used herein, oligosaccharide refers to a disaccharide to decasaccharide formed by the linkage of 2 to 10 monosaccharides. The oligosaccharide produced in this embodiment is preferably selected from the group consisting of disaccharides to octasaccharides, more preferably selected from the group consisting of disaccharides to hexasaccharides, and even more preferably selected from the group consisting of disaccharides to pentasaccharides.

[0026] The oligosaccharides to be selected are not particularly limited as long as they can be produced by a Salarria yeast or glycosyltransferase, but examples include disaccharides (e.g., sucrose, lactose, trehalose, maltose, isomaltose, cellobiose, melibiose, and gentiobiose), trisaccharides (e.g., kestose, neokestose, raffinose, panose, isopanose, maltotriose, melezitose, gentianose, lactosucrose, galactosyllactose, isomaltotriose, and gentiotriose), and tetra- to decasaccharide sugars (e.g., nystose, fructofuranosylnystose, stachyose, nigerotetraose, and gentiotetraose), as well as mixtures thereof. A mixture of oligosaccharides containing at least one selected from the group consisting of disaccharides, trisaccharides, and tetra- to decasaccharide sugars is preferred. In such a mixture, the disaccharides, trisaccharides, and tetra- to decasaccharide sugars may be present in any proportion. Furthermore, disaccharides may contain one or more types of disaccharides in any ratio, trisaccharides may contain one or more types of trisaccharides in any ratio, and sugars from tetrasaccharides to decasaccharides may contain one or more types of sugars in any ratio.

[0027] In the method of this embodiment, a single type of oligosaccharide may be selected as the oligosaccharide to be produced, but a mixture of multiple types of oligosaccharides selected from disaccharides, trisaccharides, and tetrasaccharides or larger sugars is preferably selected. Specific examples of such oligosaccharides include fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, and gentiooligosaccharides. Among these, fructooligosaccharides, isomaltooligosaccharides, and gentiooligosaccharides are preferred, with fructooligosaccharides and isomaltooligosaccharides being more preferred. Here, the gentiooligosaccharide may be a glucooligosaccharide containing gentiobiose. The oligosaccharide to be produced preferably contains an oligosaccharide of three or more sugars.

[0028] Therefore, selecting oligosaccharides to be produced from multiple types of oligosaccharides may mean selecting one or more oligosaccharides to be produced from fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, and gentiooligosaccharides, or may mean selecting one or more oligosaccharides to be produced from fructooligosaccharides, isomaltooligosaccharides, and gentiooligosaccharides, or may mean selecting one or more oligosaccharides to be produced from fructooligosaccharides and isomaltooligosaccharides. Here, the gentiooligosaccharide may be a glucooligosaccharide containing gentiobiose.

[0029] Here, fructooligosaccharides are oligosaccharides mainly containing kestose, nystose, and fructofuranosylnystose, galactooligosaccharides are oligosaccharides mainly containing galactosyllactose, isomaltooligosaccharides are oligosaccharides mainly containing isomaltose, isomaltotriose, and panose, and gentiooligosaccharides are oligosaccharides mainly containing gentiobiose, gentiotriose, and gentiotetraose.

[0030] (Carbon source selection) The method of this embodiment includes selecting one or more carbon sources from multiple carbon sources depending on the selected oligosaccharide. The carbon source to be selected is not particularly limited as long as it is a compound that can be assimilated by the yeast of the genus Salaria or that can be transglycosylated by a glycosyltransferase. Examples include disaccharides such as sucrose, lactose, trehalose, maltose, isomaltose, melibiose, and cellobiose; monosaccharides such as erythritol, xylose, galactose, glucose, fructose, and mannose; and mixtures thereof. The various carbon sources may be contained in any proportion. Furthermore, the disaccharide may contain one or more disaccharides in any proportion, and the monosaccharide may contain one or more monosaccharides in any proportion.

[0031] In the method of the present embodiment, one type of carbon source may be selected as the carbon source to be produced, or a mixture of multiple types of carbon sources selected from monosaccharides and disaccharides may be selected, or a mixture of multiple types of carbon sources selected from disaccharides may be selected.

[0032] In the method of this embodiment, the one or more carbon sources are preferably selected from monosaccharides or disaccharides, more preferably from disaccharides, and even more preferably from sucrose, maltose, and cellobiose. Here, a mixture containing monosaccharides and disaccharides in any ratio may be selected, a mixture containing multiple disaccharides in any ratio may be selected, or a mixture containing one or more selected from the group consisting of sucrose, maltose, and cellobiose in any ratio may be selected.

[0033] The carbon source is selected depending on the selected oligosaccharide. Specifically, a carbon source necessary for at least one selected from the group consisting of a Zararia yeast, a glycosyltransferase derived from a Zararia yeast, and a transformant transformed to express the glycosyltransferase to produce the selected oligosaccharide is selected. The carbon source corresponding to the selected oligosaccharide may be identified using a Zararia yeast or the like, as appropriate. For example, when the selected oligosaccharide contains a fructooligosaccharide, at least sucrose may be selected as the carbon source. When the selected oligosaccharide contains an isomaltooligosaccharide, at least maltose may be selected as the carbon source. When the selected oligosaccharide contains a gentiooligosaccharide, at least cellobiose may be selected as the carbon source. Here, the gentiooligosaccharide may be a glucooligosaccharide containing gentiobiose.

[0034] Furthermore, the correspondence between oligosaccharides and carbon sources can be determined by contacting at least one species selected from the group consisting of Zararia yeast, glycosyltransferases derived from Zararia yeast, and transformants transformed to express glycosyltransferases with an appropriate carbon source and then confirming the oligosaccharides produced.

[0035] (Oligosaccharide production) The method of this embodiment includes contacting a selected carbon source with at least one selected from the group consisting of a Zararia yeast, a glycosyltransferase derived from a Zararia yeast, and a transformant transformed to express the glycosyltransferase. In this contacting step, the at least one selected from the group consisting of a Zararia yeast, a glycosyltransferase derived from a Zararia yeast, and a transformant transformed to express the glycosyltransferase produces a corresponding oligosaccharide from the selected carbon source. Specifically, by using at least one selected from the group consisting of a Zararia yeast, a glycosyltransferase derived from a Zararia yeast, and a transformant transformed to express the glycosyltransferase, the selected carbon source undergoes a glycosyltransferase reaction, and a sugar (or a portion thereof) used as the carbon source is transferred to another sugar, thereby producing an oligosaccharide.

[0036] When at least one of a yeast of the genus Zararia and a transformant is used in the contacting step, the contacting step may include culturing at least one of a yeast of the genus Zararia and a transformant in the presence of a selected carbon source. When a glycosyltransferase is used in the contacting step, the contacting step may include producing an oligonucleotide from the selected carbon source in the presence of a glycosyltransferase derived from a yeast of the genus Zararia.

[0037] The conditions for culturing the bacterial cells can be appropriately selected depending on the culture product. For example, when culturing a yeast of the genus Zararia, a selected carbon source and the yeast of the genus Zararia may be added to an appropriate medium and cultured at 20 to 50° C., preferably 25 to 40° C. Any conventionally known growth medium may be used as the medium.

[0038] The medium used for culturing the yeast of the genus Zararia may contain an organic carbon source and / or an inorganic carbon source other than the carbon source selected above. Examples of organic carbon sources include carbohydrates such as starch and starch hydrolysates, plant cell wall components such as cellulose, xylan, and pectin, and hydrolysates thereof, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol. Inorganic carbon sources include, for example, carbon dioxide, carbonate ions, bicarbonate ions, and carbon monoxide. Carbonate ions and bicarbonate ions may be added to the medium as metal salts. Carbon dioxide and carbon monoxide may also be bubbled.

[0039] The medium used for culturing Salararia yeast may contain a nitrogen source, such as ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, inorganic or organic nitrates such as ammonium nitrate, sodium nitrate, and potassium nitrate, other nitrogen-containing compounds, as well as peptone, polypeptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean and soybean hydrolysate, soybean meal and soybean meal hydrolysate, pea and pea hydrolysate, various fermentation bacteria and digests thereof, etc. One or more of each of the organic carbon source, inorganic carbon source, and nitrogen source may be used. Suitable media include, for example, YPD medium and YPDA medium containing yeast extract and polypeptone.

[0040] When culturing a transformant, a selected carbon source and the transformant may be added to an appropriate medium, and the transformant may be cultured at a temperature appropriate for culturing the transformant (e.g., 20 to 50°C or 25 to 40°C). Any conventionally known growth medium may be used as the medium.

[0041] The medium used to culture the transformant may contain at least one of an organic carbon source, an inorganic carbon source, and a nitrogen source other than the carbon source selected above. Examples of the organic carbon source, inorganic carbon source, and nitrogen source may be the same as those exemplified for the culture medium for the yeast of the genus Salararia.

[0042] When reacting a carbon source with a glycosyltransferase, the selected carbon source and glycosyltransferase are added to an appropriate solution and incubated at the optimal temperature for the glycosyltransferase (e.g., 10 to 60°C or 25 to 50°C). The solution may be a buffer solution whose pH is maintained within an appropriate range (e.g., 4.0 to 8.0), such as a sodium acetate buffer solution.

[0043] The concentrations of the selected carbon sources during cultivation of the Zararia yeast and / or the transformant are not particularly limited, but may each independently be 1 to 50% by mass, preferably 3 to 40% by mass or 5 to 30% by mass. The total concentration of the selected carbon sources is not particularly limited, but may be 1 to 60% by mass, preferably 3 to 40% by mass or 5 to 30% by mass.

[0044] When reacting carbon sources with glycosyltransferases, the concentrations of the selected carbon sources are not particularly limited, but may each independently be 5 to 80% by mass, preferably 10 to 75% by mass or 20 to 70% by mass. The total concentration of the selected carbon sources is not particularly limited, but may be 5 to 90% by mass, preferably 10 to 80% by mass or 20 to 70% by mass.

[0045] The culture time may be adjusted appropriately depending on the rate of oligosaccharide production, and may be, for example, 1 to 150 hours, 5 to 120 hours, 10 to 100 hours, 12 to 90 hours, 20 to 85 hours, or 24 to 80 hours.

[0046] The culture can be carried out under a suitable atmosphere, for example, under CO2 conditions or atmospheric conditions. The amount of at least one selected from the group consisting of a Zararia yeast, a glycosyltransferase derived from a Zararia yeast, and a transformant transformed to express a glycosyltransferase during culture can be appropriately adjusted depending on the amount of oligosaccharides to be produced and the amount of the selected carbon source to be added.

[0047] One aspect of the method of this embodiment is a method for producing isomaltooligosaccharides, comprising producing isomaltooligosaccharides by contacting maltose with at least one yeast selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express a glycosyltransferase. In such a production method, sucrose may be present together with maltose, and fructooligosaccharides may be produced in addition to isomaltooligosaccharides. Alternatively, cellobiose may be present together with maltose, and an oligosaccharide containing gentiobiose in addition to isomaltooligosaccharides, such as gentiooligosaccharides, may be produced. Alternatively, sucrose and cellobiose may be present together with maltose, and an oligosaccharide containing fructooligosaccharides and gentiobiose in addition to isomaltooligosaccharides, such as gentiooligosaccharides, may be produced.

[0048] One aspect of the method of this embodiment is a method for producing oligosaccharides, comprising producing gentiobiose-containing oligosaccharides, for example, gentiooligosaccharides, by contacting cellobiose with at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express a glycosyltransferase. In such a production method, sucrose may be present together with cellobiose, and fructooligosaccharides may be produced in addition to the gentiobiose-containing oligosaccharides. Alternatively, maltose may be present together with cellobiose, and isomaltooligosaccharides may be produced in addition to the gentiobiose-containing oligosaccharides. Alternatively, sucrose and maltose may be present together with cellobiose, and fructooligosaccharides and isomaltooligosaccharides may be produced in addition to the gentiobiose-containing oligosaccharides.

[0049] [Oligosaccharide production system] Another aspect of this embodiment is an oligosaccharide production system that includes at least one selected from the group consisting of a Zararia yeast, a glycosyltransferase derived from a Zararia yeast, and a transformant transformed to express the glycosyltransferase, and that is capable of producing two or more types of oligosaccharides by adding one or more carbon sources selected from multiple types of oligosaccharides according to the type of oligosaccharide selected from the multiple types of oligosaccharides, and producing two or more types of oligosaccharides corresponding to the added carbon sources.

[0050] The oligosaccharide production system includes at least one selected from the group consisting of a Zararia yeast, a glycosyltransferase derived from a Zararia yeast, and a transformant transformed to express the glycosyltransferase. The Zararia yeast, the glycosyltransferase derived from a Zararia yeast, and the transformant may be the same as those described above, and therefore further description will be omitted.

[0051] The oligosaccharide production system may further include an appropriate medium and solution. The medium and solution used for culturing the Zararia yeast, the glycosyltransferase derived from the Zararia yeast, and the transformant may be the same as those described above, and therefore further description thereof will be omitted.

[0052] One or more carbon sources are added to the oligosaccharide production system, and the oligosaccharide production system produces oligosaccharides corresponding to the carbon sources. Thus, in using the oligosaccharide production system, the oligosaccharides to be produced are selected from multiple types of oligosaccharides, and the carbon source is selected according to the type of oligosaccharide selected.

[0053] The selected and produced oligosaccharides may be the same as those described above, and therefore, further explanation is omitted. Furthermore, the carbon source selected corresponding to the oligosaccharide and the relationship between the carbon source and the oligosaccharide may also be the same as those described above, and therefore, further explanation is omitted.

[0054] The oligosaccharide production system may continuously add one or more carbon sources to continuously produce the corresponding oligosaccharides, or may add one or more carbon sources batchwise to intermittently produce the corresponding oligosaccharides. Furthermore, after one or more carbon sources are selected for an oligosaccharide production system, only the selected carbon sources may be added. Alternatively, one or more selected carbon sources may be added a first time to produce the corresponding oligosaccharides, and then one or more selected carbon sources may be added a second time to produce the corresponding oligosaccharides. That is, the oligosaccharide production system may continue to add the one or more carbon sources selected and added initially, or may stop adding the one or more carbon sources selected and added initially, stop the production of oligosaccharides, and then add one or more carbon sources different from those selected and added initially.

[0055] One aspect of the oligosaccharide production system of this embodiment is a system for producing isomaltooligosaccharides from maltose, comprising at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express the glycosyltransferase. In such a system, sucrose may be added together with maltose to produce fructooligosaccharides in addition to isomaltooligosaccharides. Alternatively, cellobiose may be added together with maltose to produce oligosaccharides containing gentiobiose in addition to isomaltooligosaccharides, such as gentiooligosaccharides. Alternatively, sucrose and cellobiose may be added together with maltose to produce oligosaccharides containing fructooligosaccharides and gentiobiose in addition to isomaltooligosaccharides, such as gentiooligosaccharides.

[0056] One aspect of the oligosaccharide production system of this embodiment is a system that produces gentiobiose-containing oligosaccharides, such as gentiooligosaccharides, from cellobiose, and includes at least one yeast selected from the group consisting of a Salararia yeast, a glycosyltransferase derived from a Salararia yeast, and a transformant transformed to express the glycosyltransferase. In such a system, sucrose may be added together with cellobiose to produce fructooligosaccharides in addition to the gentiobiose-containing oligosaccharides. Alternatively, maltose may be added together with cellobiose to produce isomaltooligosaccharides in addition to the gentiobiose-containing oligosaccharides. Alternatively, sucrose and maltose may be added together with cellobiose to produce fructooligosaccharides and isomaltooligosaccharides in addition to the gentiobiose-containing oligosaccharides.

[0057] [Separation and use of oligosaccharides] The oligosaccharide solution (reaction solution containing oligosaccharides) produced by the method or system of this embodiment may be used as a composition containing oligosaccharides, or the oligosaccharides may be separated from the oligosaccharide solution. Methods for separating oligosaccharides include one or a combination of methods selected from the group consisting of chromatography (e.g., ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography), membrane separation, solvent precipitation, activated carbon, and adsorbents.

[0058] The obtained oligosaccharide liquid or oligosaccharides may be used as liquid sugar or powdered using a spray dryer, freeze dryer, etc. The oligosaccharide liquid or oligosaccharides may be used in a variety of applications, such as food additives such as sweeteners, taste improvers, stabilizers, discoloration inhibitors, and excipients, as well as cosmetics, pharmaceuticals, feed, and industrial products. [Example]

[0059] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples. Those skilled in the art can modify the present invention in various aspects without departing from the spirit of the present invention, and such modifications are also within the scope of the present invention.

[0060] As Zararia yeast, Zalaria Him3 (see Non-Patent Document 1). Yeast extract polypeptone dextrose agar medium (YPDA medium) (yeast extract 10 g / L, polypeptone 5 g / L, glucose 20 g / L, agar 20 g / L) was used. Zalaria It was used to maintain sp. Him3. In the following, oligosaccharides were identified by high performance liquid chromatography (HPLC). For HPLC conditions, see Non-Patent Document 1.

[0061] Example 1 Zalaria For the preculture of sp. Him3, a loopful of YPDA culture was inoculated into a test tube containing 3 mL of YPD liquid medium and incubated at 30°C for 1 day with reciprocal shaking at 180 rpm. One mL of this preculture was inoculated into an Erlenmeyer flask containing 100 mL of 15% sucrose liquid medium (15% sucrose by weight, 10 g / L yeast extract, 5 g / L polypeptone) and incubated at 30°C with rotary shaking at 200 rpm. The culture solution was analyzed at 24, 48, and 72 hours after the start of incubation by thin-layer chromatography (TLC) using n-butanol / methanol / water (5:3:2, v / v / v) as the developing solvent. Oligosaccharide production was confirmed by spraying with 30% sulfuric acid and then heating at 100°C. The results are shown in Figure 1. After 24 hours of incubation, spots of monosaccharides and sucrose, as well as spots of oligosaccharides, were observed. After 48 and 72 hours of incubation, the monosaccharides and sucrose decreased, and spots of oligosaccharides with a higher degree of polymerization were observed. Furthermore, this oligosaccharide fraction contained fructooligosaccharides.

[0062] <Example 2> The test was carried out in the same manner as in Example 1, except that maltose was used in place of sucrose in the medium. The results are shown in Figure 1. After 24 hours of incubation, a maltose spot was observed. After 48 and 72 hours of incubation, the amount of maltose decreased, and oligosaccharide spots were observed. This oligosaccharide fraction also contained isomaltooligosaccharides.

[0063] Example 3 The test was carried out in the same manner as in Example 1, except that cellobiose was used at 10% by mass in place of sucrose in the medium. The results are shown in Figure 2. From the left, the results are for a cellobiose preparation, and for 24 and 48 hours after the start of incubation. After 24 hours of incubation, a cellobiose spot was observed. After 48 hours of incubation, cellobiose decreased and an oligosaccharide spot was observed. This oligosaccharide fraction also contained gentiobiose.

[0064] Example 4 Zalaria Instead of sp. Him3, Zalaria It was confirmed that oligosaccharides could be produced even when glycosyltransferase extracted from sp. Him3 was used. Zalaria The Him3 strain was cultured for 2 days under the same conditions as in Example 1. The culture was centrifuged at 20,000 × g for 3 minutes at 4°C, and the supernatant was used as extracellular glycosyltransferase (EC-FFase). The precipitated cells were resuspended in the same volume of 50 mmol / L phosphate buffer (pH 7.0) as the supernatant and used as cell surface glycosyltransferase (CS-FFase). The glycosyltransferase activity of EC-FFase and CS-FFase was evaluated by measuring the amount of glucose produced from sucrose. One unit (U) of glycosyltransferase activity was defined as the amount of enzyme that releases 1 μmol of glucose per minute.

[0065] 10 U of CS-FFase or EC-FFase was added to a reaction mixture (10 mL) prepared by dissolving 500 g / L sucrose in 100 mmol / L sodium acetate buffer (pH 5.0), and the mixture was incubated at 30° C. The results showed that fructooligosaccharides were produced when either CS-FFase or EC-FFase was used.

Claims

1. A method for producing oligosaccharides, comprising: selecting an oligosaccharide to be produced from a plurality of types of oligosaccharides; selecting one or more carbon sources from a plurality of carbon sources according to the type of oligosaccharide selected; producing the selected oligosaccharide by contacting the selected carbon source with at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express the glycosyltransferase; A method comprising:

2. The oligosaccharides produced are selected from fructooligosaccharides, isomaltooligosaccharides, and gentiooligosaccharides. The method of claim 1.

3. the one or more carbon sources are selected from monosaccharides or disaccharides; 3. The method according to claim 1 or 2.

4. The selected carbon source comprises at least one selected from sucrose, maltose, and cellobiose; 3. The method according to claim 1 or 2.

5. When the selected oligosaccharides include fructooligosaccharides, at least sucrose is selected as the carbon source; When the selected oligosaccharides include isomaltooligosaccharides, at least maltose is selected as the carbon source; When the selected oligosaccharides include gentiooligosaccharides, at least cellobiose is selected as the carbon source.

3. The method according to claim 1 or 2.

6. The yeast comprises at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express the glycosyltransferase; one or more carbon sources selected from a plurality of types of oligosaccharides according to the type of oligosaccharide selected are added to produce the type of oligosaccharide corresponding to the added carbon source; An oligosaccharide production system capable of producing two or more types of oligosaccharides.

7. A method for producing isomaltooligosaccharides, comprising producing isomaltooligosaccharides by contacting maltose with at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express the glycosyltransferase.

8. The yeast comprises at least one selected from the group consisting of a yeast of the genus Zararia, a glycosyltransferase derived from a yeast of the genus Zararia, and a transformant transformed to express the glycosyltransferase; Producing isomaltooligosaccharides from maltose Isomaltooligosaccharide production system.

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