Aldose production method

Abstract

This invention relates to a technology that can more efficiently produce rare sugars, particularly aldohexoses, using inexpensive sugars as raw materials. Specifically, it involves producing a particular rare sugar using the cells of a particular microorganism or a dehydrogenase that oxidizes the sugar alcohol produced by that microorganism in the presence of a specific substrate sugar alcohol. [Solution] The solution involves using D-sorbitol, L-sorbitol, L-iditol, L-talitol, and allitol as substrates and a specific Ps-ADH enzyme to produce L-growth, D-growth, L-idose, L-altrose, and L-allose.

Description

Technical Field

[0001] The present invention relates to a technology capable of more efficiently producing rare sugars in aldoses, particularly aldohexoses, using inexpensive sugars as raw materials. Specifically, it involves producing specific rare sugars using dehydrogenases that oxidize the cells of specific microorganisms or the sugar alcohols produced by said bacteria in the presence of a sugar alcohol serving as a specific substrate.

Background Art

[0002] Aldose is a name used when classifying carbohydrates by their structure. It has one formyl group at the end of the chain and refers to monosaccharides with the chemical formula C n H 2n O n (n≧3). And aldohexose is an organic compound containing six carbon atoms and a formyl group.

[0003] Aldohexoses such as D-glucose are monosaccharides with six carbon atoms and are classified into a total of 16 types of aldohexoses according to the orientation of multiple hydroxyl groups. Among them, the three types that are commonly found in nature, sold at low prices, and easily available are D-glucose, D-galactose, and D-mannose. The remaining aldohexoses are classified as rare sugars, with high prices and difficult availability.

[0004] Recently, rare sugars have a chemical structure of a sugar skeleton that is considered to be highly safe for organisms, and unlike glucose, they have the property of being less metabolized by living organisms. Therefore, they are considered useful in fields such as sweeteners. In addition, some rare sugars have notable physiological activities and are recognized and attracting attention for their usefulness in the technical field of the present invention. Thus, rare sugars are recognized as being highly industrially applicable and effective.

[0005] One known technique for producing aldohexoses, which are classified as rare sugars, involves using an isomerase such as rhamnose isomerase to convert ketoses, including D-fructose, into aldoses (Non-Patent Literature 1). This enzyme converts ketoses into aldoses by converting the ketone group at the C-2 position of the ketose to a hydroxyl group, and further converting the adjacent hydroxymethyl group to an aldehyde group.

[0006] Several methods for producing aldoses from sugar alcohols are known, including the conversion reaction of D-sorbitol to L-glucose and D-mannitol to mannose using polyol oxidase derived from Penicillium bacteria (Patent Document 1). Another known method involves using polyol oxidase derived from microorganisms belonging to the Arthrobacter genus to oxidize one of the hydroxyl groups at both ends of D-sorbitol, L-talitol, galactitol, D-mannitol, and D-talitol to produce D-glucose, L-altrose, D-galactose, D-mannose, and D-talose, respectively (Patent Document 2).

[0007] However, as mentioned above, although various methods for producing rare sugars are known, the production of most rare sugars remains difficult, and they are traded at extremely high prices. In particular, D-growth, L-allose, L-altrose, and L-idose are extremely expensive and difficult to obtain, and L-growth is also difficult to acquire. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2013 / 062102 [Patent Document 2] Patent application 2016-226803 (Patent No. 6856894) [Patent Document 3] Japanese Patent Application Publication No. 5-68542 [Patent Document 4] International Publication No. 2008 / 139844 [Patent Document 5] International Publication No. 2013 / 183610 [Non-patent literature]

[0009] [Non-Patent Document 1] Chemistry and Biology Vol. 56, pp. 752-758, 2018 [Non-Patent Document 2] Biosci. Biotechnol. Biochem. 86,56-67(2021) [Non-Patent Document 3] Appl Biochem Biotechnol. 196,1876-1895(2024) [Overview of the project] [Problems that the invention aims to solve]

[0010] As mentioned above, D-growth, L-allose, L-altrose, L-idose, and L-growth are extremely difficult to produce and mass-produce, and therefore there are few documents describing their usefulness. However, they are low in calories, yet exhibit a sweetness similar to glucose, and are also highly safe. There is high demand for them among those skilled in the art. Furthermore, these rare sugars are highly likely to possess beneficial physiological activity. Therefore, these rare sugars have high industrial applicability and usefulness, and there is significant demand from those skilled in the art.

[0011] However, as mentioned above, although several methods for producing rare sugars have been proposed, for various reasons, most rare sugars are currently sold at very high prices. In particular, D-growth, L-allose, L-althrose, and L-idose are extremely expensive, and L-growth is also an expensive carbohydrate. Therefore, in order to meet the strong demand for these carbohydrates, there is a need to develop a technology that can produce them more efficiently using cheaper sugars as raw materials. This is the main problem that the present invention aims to solve. [Means for solving the problem]

[0012] To solve the above problems, the inventors of this application diligently investigated bacteria, substances, methods, and conditions that could satisfy these requirements. As a result, they found that a special alcohol dehydrogenase described in Non-Patent Document 2 (hereinafter sometimes referred to as Ps-ADH), which is homologous to conventionally existing alcohol dehydrogenases, can be used.

[0013] Furthermore, Ps-ADH oxidizes D-sorbitol (Patent Document 3). Theoretically, the oxidation of D-sorbitol should produce not only D-glucose but also L-glucose. However, according to Non-Patent Document 3, the activity of this enzyme towards L-glucose is very low, about 1 / 20 or less of that towards D-glucose, so the production of L-glucose during D-sorbitol oxidation has been questioned. This is, so to speak, the conventional technical common sense in this industry.

[0014] However, despite this common technical knowledge, the inventors conducted experiments such as the analysis of reaction products during the oxidation of D-sorbitol. As a result, they found that while the main product of the oxidation reaction of D-sorbitol is D-glucose, L-glucose can also be produced. This is one of the present inventions.

[0015] Furthermore, the oxidative activity of Ps-ADH towards sorbitol is as high as that of D-glucose, one of the best substrates. Therefore, in conjunction with the known oxidation reaction pathway for D-glucose, it is presumed that this enzyme catalyzes the conversion reactions of D-sorbitol, D-glucose, D-glucaraldehyde, and D-glucuronic acid (Non-Patent Literature 2).

[0016] In addition, the inventors of the present invention added a commercially available glucose dehydrogenase that specifically oxidizes the produced D-glucose, or reacted resting cells of Ps-ADH, Pseudogluconobacter saccharoketogenes Rh-47-3 strain, or Yohiella xiaoguannensis JCM30821 strain for a long time (about 24 hours) to selectively or preferentially oxidize D-glucose to convert it into an acidic sugar lactone (D-glucono-1,5-lactone), and succeeded in leaving only L-glucose as a neutral sugar.

[0017] D-glucose and L-glucose have the same structural formula, and the difference is only in the orientation of the hydroxyl group. Therefore, it is difficult to separate and purify both of them while remaining as neutral sugars. However, if D-glucose is converted into an acidic sugar as described above, it becomes possible to easily separate D-glucose and L-glucose using an anion exchange column or the like.

[0018] In the present invention, Pseudogluconobacter saccharoketogenes can be used. This bacterium is known to oxidize the C-6 position of D-glucose to produce uronic acids such as D-glucuronic acid and L-glucuronic acid (Patent Document 4), and further oxidize these uronic acids to produce D-glucaric acid (Patent Document 5, Non-Patent Document 2). The enzyme that oxidizes the C-6 position of this bacterium is the above-mentioned Ps-ADH.

[0019] In addition, although the enzyme has not been identified, it has been described that this bacterium shows oxidation activity against sugar alcohols derived from aldohexoses such as D-sorbitol, D-mannitol, and galactitol (Patent Document 3). When the sugar alcohol is oxidized, there are cases where an aldose in which the C-1 position is oxidized to an aldehyde group is generated, but there are also many cases where a ketose in which the C-2 position becomes a ketone is generated. Furthermore, depending on which of the hydroxymethyl groups at both ends of D-sorbitol or D-galactitol is oxidized, there is a possibility of generating two types of aldoses, namely D-glucose or L-glucose and D- or L-galactose, respectively. However, these documents do not mention the products formed during the oxidation of these sugar alcohols, and the sugars produced are unknown. In this technical field, it is common for what is theoretically conceivable to differ from what actually occurs, and often it is not known whether the expected effects will be achieved without actually trying.

[0020] The present invention has been made under such a technical background, and has identified the substrate specificity of Ps-ADH for sugar alcohols and the products formed during oxidation, and has found that this enzyme oxidizes these sugar alcohols to produce the corresponding aldoses.

[0021] The present invention has finally been completed as a result of further research based on such useful new findings. Hereinafter, the present invention will be described in detail.

[0022] First, the main embodiments of the present invention are as follows. (1) A method for producing L-glucose, characterized by separating and obtaining L-glucose produced by using a dehydrogenase that oxidizes sugar alcohols of a microorganism belonging to the genus Pseudogluconobacter, genus Yohiella, genus Paradevosia, genus Methyloterrigena or genus Devosia or the same bacterium in the presence of D-sorbitol. (Production method). (2) A method for producing D-glucose, characterized by separating and obtaining D-glucose produced by using a dehydrogenase that oxidizes sugar alcohols of the microorganism cells or the same microorganism-derived sugar alcohols described in (1) in the presence of L-sorbitol. (Production method). (3) A method for producing L-idose, characterized by producing it by using a dehydrogenase that oxidizes sugar alcohols of the microorganism cells or the same microorganism-derived sugar alcohols described in (1) in the presence of L-iditol. (Production method). (4) A method for producing L-altrose, characterized by producing it by using a dehydrogenase that oxidizes sugar alcohols of the microorganism cells or the same microorganism-derived sugar alcohols described in (1) in the presence of L-talitol (L-altritol). (Production method). (5) A method for producing L-allose, characterized by producing it in the presence of allitol using a dehydrogenase that oxidizes the microbial cells described in (1) or a sugar alcohol derived from the same microorganism. (Production method). [Effects of the Invention]

[0023] The manufacturing method according to the present invention has the remarkable effect of enabling the inexpensive and efficient production of rare sugars, which were previously very expensive. In particular, D-growth, L-allose, L-althrose, L-idos, and L-growth can be produced efficiently using less expensive sugars as raw materials.

[0024] These rare sugars, while also carbohydrates, differ from glucose in that they are absorbed more slowly by the body, making them less likely to cause excessive carbohydrate intake. Despite this, they still provide sufficient sweetness for human perception, and some are comparable to sucrose and other sugars. Furthermore, the functional properties of these rare sugars in terms of physiological activity have also been studied and are very promising. Thus, rare sugars are substances whose production is highly desired in many industries, and this invention, which can meet this demand, is extremely useful. [Brief explanation of the drawing]

[0025] [Figure 1] This figure shows a sugar alcohol that serves as a substrate and the aldose produced, which are part of the present invention. [Figure 2]This shows the chromatogram of the reaction solution obtained by oxidizing sugar alcohols with Ps-ADH in Example 2, using high-performance liquid anion chromatography (HPAEC-PAD) with a pulsed amperometry detector. D-sorbitol, which oxidizes efficiently, was reacted at 125 mM, while the other sugar alcohols were reacted at 12.5 mM. The reaction solutions obtained by oxidizing D-sorbitol (A), L-sorbitol (B), L-iditol (C), L-talitol (D), and allitol (E) with Ps-ADH were analyzed using HPAEC-PAD. Simultaneously, standards of various aldoses were analyzed to identify the aldoses produced when various sugar alcohols are oxidized. [Figure 3] This figure shows the HPAEC-PAD analysis of the reaction mixture obtained when various sugar alcohols were oxidized using resting cells of the Rh47-3 strain in Example 3. Each sugar alcohol was reacted in a reaction mixture containing resting cells of the Rh47-3 strain. The aldose produced and the substrate sugar alcohol are shown in the figure. [Figure 4] This figure shows the HPAEC-PAD analysis of the reaction solutions obtained when various sugar alcohols were oxidized using resting cells of the JCM30821 strain in Example 3. Each sugar alcohol was reacted in a reaction solution containing resting cells. The aldose produced and the substrate sugar alcohol are shown in the figure. [Figure 5] This figure shows the HPAEC-PAD analysis of the reaction solutions obtained when various sugar alcohols were oxidized using resting cells of the JCM19854 strain in Example 3. Each sugar alcohol was reacted in a reaction solution containing resting cells. The aldose produced and the substrate sugar alcohol are shown in the figure. [Figure 6] This document describes the identification of D-glucose produced during D-sorbitol oxidation in Example 3. Solutions of D-sorbitol oxidized in resting cells of Rh47-3 and JCM30821 were treated with the enzyme solution provided with the Glucose CII-Test Wako (Glc test+) and untreated solutions (Blank), and then subjected to HPAEC-PAD analysis. [Figure 7]This shows the identification of allose produced during the oxidation of alitol in Example 4. Ps-ADH oxidizes D-allose (D-allose standard) but does not oxidize L-allose (L-allose standard). When the alitol oxidation reaction solution was treated with Ps-ADH, the peak area of ​​L-allose hardly changed before and after enzymatic treatment, indicating that L-allose is the main product of the alitol oxidation reaction. [Figure 8] The gene sequence of Ps-ADH from Yohaiella shaoguanensis JCM38021 is shown. [Figure 9] The amino acid sequence of Ps-ADH from Yohaiella shaoguanensis JCM38021 is shown. [Figure 10] The gene sequence of Ps-ADH from Yojayella tivetensis JCM19854 is shown. [Figure 11] The amino acid sequence of Ps-ADH from Yojayella tivetensis JCM19854 is shown. [Modes for carrying out the invention]

[0026] The present invention will be described in detail below.

[0027] The present invention relates to a method for producing aldoses, characterized by reacting sugar alcohols and their derivatives having a hydroxymethyl group, or polysaccharides, prepared as raw materials, with microorganisms belonging to the genera Pseudogluconobacter, Yojayella, Paradevosia, Methyloteligna, or Devosia, or their processed products, which have the ability to oxidize the hydroxymethyl group to an aldehyde group, thereby generating and accumulating the corresponding aldose, and collecting it.

[0028] It has been revealed that Ps-ADH, the enzyme used in this invention, is unique in that it does not have homology with any known dehydrogenase or any other enzyme (Non-Patent Literature 2).

[0029] The inventors searched and investigated whether there were any microorganisms capable of preparing aldoses from sugar alcohols, similar to the resting cells of Pseudogluconobacter saccharoketogenes used in the present invention. As a result, we also found that the cells of Yojayella xiaoguanensis JCM30821 and Yojayella tibetensis JCM19854 can produce the corresponding rare sugars from sugar alcohols, similar to Ps-ADH.

[0030] Furthermore, proteins derived from the genus Devosia H5989 and Devosia SP. D6-9 were also identified. In particular, the protein derived from Devosia H5989 showed 99.6% gene sequence homology and 100% amino acid sequence homology with Ps-ADH from Yojayella tivetensis JCM19854 when the sequence was analyzed. This Ps-ADH is also considered to reliably catalyze the reaction of the present invention.

[0031] When the amino acid sequences of the four proteins mentioned above and Ps-ADH were examined using the gene database (NCBI BLAST http: / / www.ncbi.n1m.nih.gov / BLAST / ), they were found to have more than 90% homology. It is believed that the fact that these four proteins catalyze the reaction of the present invention is not publicly known.

[0032] The series of reactions in the method for producing aldose from sugar alcohols is shown in Figure 1. The sugar alcohols used in this invention can be aldoses, ketoses or their derivatives synthesized by chemical reactions, enzymes or microbial cells, sugar alcohols chemically synthesized from other raw materials, or sugar alcohols extracted from natural products. These can be selected appropriately after considering the cost and manufacturing process.

[0033] The microorganisms belonging to the genera Pseudogluconobacter, Yojayella, Paradevosia, Methyloteligna, or Devosia that can be used in the present invention may be any microorganism belonging to the genera Pseudogluconobacter, Yojayella, Paradevosia, Methyloteligna, or Devosia that have the ability to oxidize the hydroxymethyl group at the C-1 position of a sugar alcohol to an aldehyde group, and include mutant strains obtained by conventional mutagenesis operations, such as treatment with mutagens like nitrosoguanidine, ultraviolet irradiation, or genetic recombination.

[0034] Of the microorganisms listed above, Pseudogluconobacter saccharoketogenes, Yohaiella shaoguanensis, or Yohaiella tibetensis are particularly preferred. More specifically, for example, the following strains of the Pseudogluconobacter genus are suitable representative examples. Pseudogluconobacter saccharoketogenes K591s strain: FERM BP-1130, IFO 14464. Pseudogluconobacter saccharoketogenes 12-5 strain: FERM BP-1129, IFO 14465. Pseudogluconobacter saccharoketogenes TH 14-86 strain: FERM BP-1128, IFO 14466. Pseudogluconobacter saccharoketogenes 12-15 strain: FERM BP-1132, IFO 14482. Pseudogluconobacter saccharoketogenes 12-4 strain: FERM BP-1131, IFO 14483. Pseudogluconobacter saccharoketogenes 22-3 strain: FERM BP-1133, IFO 11484.

[0035] The genus Youhaiella includes species such as Youhaiella shaoguanensis (JCM38021), also known as Paradevosia shaoguanensis or Methyloterrigena soli, and Youhaiella tibetensis (JCM19854).

[0036] Notable species in the genus Devosia include Devosia sp.D6-9 (Taxonomy ID:2664417) and Devosia sp.H5989 (D6-9, Taxonomy ID:1643450).

[0037] The microorganisms used in this invention can be any microorganism that produces an enzyme capable of selectively oxidizing the C1 position of a sugar alcohol, and for example, microorganisms belonging to the following genera can be used. Examples include genera such as Pseudogluconobacter, Yojayella, Paradevosia, Methyloteligna, and Devosia.

[0038] Among these microorganisms, the following are examples of suitable microorganisms. Pseudogluconobacter saccharoketogenes Rh47-3 (FERM BP-10820) Youhaiella shaoguanensis: Also known as Paradevosia shaoguanensis, JCM 30821 Youhaiella tibetensis (JCM 19854) Methyloterrigena soli Devosia sp. D6-9 Devosia sp. H5982

[0039] These microorganisms are either publicly known or deposited and available for distribution, and are all readily accessible. For example, strain Rh47-3 is disclosed in Patent Document 4 and has been internationally deposited as FERM BP-10820 in accordance with the so-called Budapest Convention at the Patent Organism Depositary Center of the National Institute of Technology and Evaluation (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture 292-0818). Other microorganisms are also assigned JCM numbers and GenBank numbers, so there are no difficulties in obtaining or accessing them. The JCM number refers to the Japan Collection of Microorganisms (JCM) number, assigned by the BioResource Research Center (BRC) of the RIKEN (Japan Institute of Physical and Chemical Research) (3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture 305-0074), and microorganisms are deposited and transferred using this number.

[0040] In the method according to the present invention, the cells of microorganisms belonging to the genera Pseudogluconobacter, Yojayella, Paradevosia, Methyloteligna, and Devosia may be isolated and used as is, or processed products such as cell lysates or cell cultures may be used. Furthermore, the dehydrogenase with sugar alcohol oxidation activity produced by these microorganisms, or genetically modified microorganisms in which the enzyme gene is expressed in E. coli or other organisms, or enzymes extracted from such recombinant microorganisms may be used. However, in the case of the Pseudogluconobacter, Yojayella, and Devosia microorganisms of the present invention, the enzyme is usually accumulated in the cell (periplasm). Generally, it is preferable to use the cells themselves and bring them into contact with raw sugars to produce aldoses. In particular, it is preferable to use dormant cells.

[0041] One effective method is to carry out oxidative fermentation using microorganisms such as Pseudogluconobacter saccharoketogenes. This method allows for the production of the corresponding aldose by oxidizing the hydroxymethyl group of a sugar alcohol. Specifically, as will be described later in the examples, rare sugars can be produced by applying the microorganism, genetically modified microorganism, its culture medium, or enzymes produced by the microorganism. Since fermentation by microorganisms or oxidation reactions using enzymes can be carried out under mild conditions, the desired product can be produced safely and with minimal environmental impact. Furthermore, because the desired site can be selectively oxidized, the number of steps is reduced, costs are kept low, and relatively little capital investment is required, resulting in high economic benefits.

[0042] The Ps-ADH gene can also be incorporated into a host organism such as E. coli or yeast, and gene expression can be performed to generate and utilize the enzyme from the bacterial cells or genetically modified organisms to achieve the desired activity. In this case, the nucleic acid donor for the Ps-ADH gene is not limited to the genera Pseudogluconobacter, Yojayella, Paradevosia, Methyloteligna, and Devosia, but may also be an enzyme gene from another microorganism that has more than 80% gene sequence homology to the alcohol dehydrogenase (Ps-ADH) of Pseudogluconobacter saccharoketogenes Rh47-3, Yojayella shaoguanensis JCM30821, and Yojayella tibetensis JCM19854, as shown below.

[0043] The amino acid sequence and gene sequence of Ps-ADH derived from Pseudogluconobacter saccharoketogenes Rh47-3 are described in Non-Patent Document 2.

[0044] The amino acid sequence and gene sequence (GenBank:CP068983 Region2586237-2587997) of Ps-ADH from Yohaiella shaoguanensis JCM38021 are shown in Sequence 2 and Figure 9 for the amino acid sequence, and in Sequence 1 and Figure 8 for the nucleotide sequence.

[0045] The amino acid sequence and gene sequence of Ps-ADH (GenBank:CP041690.1 Region714458-716140) from Yojayella tivetensis JCM19854 are shown in Sequence 4 and Figure 11 for the amino acid sequence, and in Sequence 3 and Figure 10 for the nucleotide sequence.

[0046] The amino acid sequence of Ps-ADH from the genus Devosia H5989 (GenBank:CP011300.1 Region4497082-4498764) was identical to that of Ps-ADH from Yojayella tivetensis JCM19854, as shown below.

[0047] We investigated the homology of the Ps-ADH gene sequence of Yojayella tivetensis JCM19854 (GenBank:CP041690.1 Region714458-716140) and the Ps-ADH gene sequence of Devosia genus H5989 (GenBank:CP011300.1 Region4497082-4498764), which has high homology with the Ps-ADH gene of Yojayella tivetensis JCM19854 (GenBank:CP041690.1 Region714458-716140). 1676 bp out of a total of 1683 bp (99.6%) of the gene sequence were identical. Furthermore, when the sequences were translated into amino acids and compared, the Ps-ADH genes of the two bacteria were identical throughout their entire length.

[0048] In the present invention, it is sufficient to prepare a reaction solution containing the corresponding aldose by oxidizing the hydroxymethyl group of a sugar alcohol by any means.

[0049] In the above reaction, water is preferred as the solvent for dissolving the substrate sugar alcohol and the product aldose. In the above reaction, the concentration of the sugar alcohol is usually 1-50%, preferably 5-30%. Furthermore, the reaction temperature is usually in the range of 0 to 60°C, preferably 15 to 40°C, but is not limited to these conditions, including the above conditions, as long as the conditions allow for efficient production. The pH of the above reaction is usually 3 to 10, preferably 4 to 8, but is not limited to these values.

[0050] Furthermore, stirring is preferable during this reaction. In particular, when the reaction is carried out with bacterial cells, it is thought that the reaction is completed when electrons extracted from the substrate sugar alcohol are transferred to oxygen via the electron transport system on the cell membrane. Therefore, stirring is preferable to create reaction conditions that efficiently supply oxygen.

[0051] The reaction time is typically 1 to 240 hours, preferably 24 to 120 hours, but is not limited to these if aldose is produced efficiently. Once the reaction has progressed and aldoses have accumulated, concentrate, decolorize, and desalt the solution. These steps can be carried out using standard methods.

[0052] The resulting reaction solution containing aldose contains salts, organic acids, colorants, and proteins derived from microorganisms, so it is best to remove these by using a resin that is acidic, basic, or both. Furthermore, activated carbon, electrodialysis equipment, etc., may be used upon request.

[0053] The following describes embodiments of the present invention, but the present invention is not limited to these embodiments, and various modifications are possible within the technical concept of the present invention. [Examples]

[0054] <method> bacterial culture and preparation of resting cells The bacteria were cultured in Ps medium (pH 7.0, containing 2% lactose, 0.5% yeast extract D-3 (Nippon Pharmaceutical Co., Ltd.), 1% corn steep liquor (Solris 095E, Oriental Yeast Co., Ltd.), 0.3% ammonium sulfate, 0.1% ferric sulfate heptahydrate, 0.01% lanthanum chloride heptahydrate, 0.001% riboflavin, and 1% calcium carbonate). Pre-culture was prepared by inoculating 1 mL of Ps medium with glycerol stock and culturing at 240 oscillations / min and 27°C for 3 days. 5 mL of Ps main culture medium was inoculated with the pre-culture to a volume of 1 / 100 of the main culture medium. The culture was carried out for 2 days under the same conditions as the pre-culture, and the cells were collected by centrifugation (10,000 × g, 5 minutes, 25°C). The cells were washed twice with physiological saline and used as resting cells.

[0055] HPAEC-PAD HPAEC-PAD was performed using a Dionex ICS-6000 (Thermo Scientific) system equipped with a Carbopak PA-10 column and an electrochemical detector. Elution was performed using a linear concentration gradient method, varying the sodium acetate concentration in 0.1 M sodium hydroxide solvent from 20 to 500 mM, with the column temperature measured at 35°C. Since the Carbopak PA-10 column used does not have chiral discrimination ability, the retention times for D- and L-isomers of the same type of aldose were exactly the same.

[0056] McKilbane Buffer McIlvaine buffer was prepared by mixing 200 mM disodium hydrogen phosphate and 100 mM citrate to a pH of 5.0 to make 1× McIlvaine buffer (pH 5.0). Diluted McIlvaine buffer pH 5.0 (×10) (Wako Pure Chemical Industries) or a prepared ×5 McIlvaine buffer (pH 5.0) solution was added to the reaction mixture to a ×1 ratio.

[0057] Partial purification of enzymes 1 mL of the pre-culture solution of the Ph47-3 strain, pre-cultured in 5 mL of Ps medium, was inoculated into 100 mL of medium in Sakaguchi flasks (5 flasks) and cultured. The cells were collected by centrifugation (5,000 × g, 15 min, 25°C) and washed twice with physiological saline. The cells were suspended in 20 mL of ×1 McIlbain buffer (pH 5.0), crushed using a French press (manufactured by Otake Seisakusho Co., Ltd.), and the crushed solution was centrifuged at 3,000 g × 15 min to collect the supernatant. Further centrifugation was performed at 5,000 g × 15 min, and the supernatant was collected again. The collected supernatant was applied to 5 mL of Hi-trap DEAE-FF (manufactured by GE Healthcare), and the enzyme was eluted using a linear concentration gradient method with a sodium chloride concentration of 0-350 mM in 10 mM phosphate buffer (pH 5.5) containing 100 mM glycerin. The fraction containing trehalose oxidation activity was recovered and used in the experiment as partially purified Ps-ADH.

[0058] Thin-layer chromatography The samples were developed using a silica gel 60 plate (Merck) with a solvent of ethyl acetate:acetic acid:water = 3:1:1. After development, the samples were thoroughly dried, then sprayed with 50% methanol sulfate, heated at 160°C, and the spots were detected.

[0059] Activity measurement A reaction mixture (100 μL) consisting of 100 mM trehalose, 1 × McIlbain buffer (pH 5.0), 20 mM potassium ferricyanide, partially purified Ps-ADH, and resting cells of each bacterial species was incubated at 40°C for 10 minutes. 50 μL of reaction stop solution (0.5% (w / v) ferric sulfate, 9.5% (v / v) phosphate) was added to stop the reaction, and 350 μL of deionized water was added. After standing in the dark for 90 minutes, the absorbance at 660 nm was measured. Enzyme activity of 1 U was defined as the amount of enzyme required to oxidize 0.5 μmol of trehalose per minute.

[0060] <result> Substrate specificity A reaction mixture (100 μL) consisting of 5 mM substrate, 1 × McIlbain buffer (pH 5.0), 20 mM potassium ferricyanide, and partially purified Ps-ADH (0.0025–0.05 U / mL) was incubated at 40°C for 10–60 minutes. Reaction termination and enzyme activity were measured using the method described above for activity measurement. Relative activity was expressed with the enzyme's oxidative activity against trehalose set to 100%. The obtained relative activities are shown in Table 1 below.

[0061] [Table 1] [Examples]

[0062] Analysis of aldoses produced when sugar alcohols are oxidized with partially purified Ps-ADH. Various sugar alcohols were reacted with partially purified Ps-ADH, and the resulting aldoses were analyzed. D-sorbitol, which has high relative oxidative activity and is inexpensive, was reacted at a substrate concentration of 125 mM, while other sugar alcohols were reacted at a substrate concentration of 12.5 mM. The reaction solution, consisting of sugar alcohol (125 mM or 12.5 mM), 1 × McIlbain buffer (pH 5.0), 4.1 mM potassium ferricyanide (12.5 mM for D-sorbitol), and 0.23 U / mL partially purified Ps-ADH, was reacted at 30°C for 3 days. After the reaction, the mixture was treated at 100°C for 5 minutes to stop the reaction, and the resulting aldoses were measured using HPAEC-PAD (Figure 2). Furthermore, because sugar alcohols have hydroxymethyl groups at both ends, theoretically, two types of aldoses can be produced depending on which end is oxidized. The sugar alcohols that may produce aldoses are also listed in Figure 2. Oxidation of D-sorbitol primarily resulted in the formation of glucose, and later L-growth (Figure 2A). On the other hand, the oxidation of L-sorbitol primarily produced D-glucose, with a small amount of L-glucose being produced (Figure 2B). L-iditol was oxidized to produce L-idose (Figure 2 C). Oxidation of L-talitol (L-althritol) can potentially produce L-talose and L-althrose, but only L-althrose was produced (Figure 2 D). The oxidation of allitol may produce D- and L-allose. While allose formation was confirmed from the HPAEC-PAD chart (Figure 2 E), the ratio of D-allose to L-allose could not be determined from the chromatogram chart because HPAEC-PAD lacks chiral discrimination capabilities. [Examples]

[0063] Identification of aldoses produced in resting cells of Rh47-3, JCM30821, and JCM19854 Residual cells of Rh47-3 (final concentration 1.28 U / mL), JCM30821 (final concentration 3.76 U / mL), and JCM19854 (final concentration 2.2 U / mL) were treated with D-sorbitol at a final concentration of 100 mM and other sugar alcohols at a final concentration of 10 mM as substrates. Furthermore, McIlbain buffer (pH 5.0) was added to a 1× ratio, and the mixture was shaken at 240 oscillations / min for 2 hours. After boiling for 5 minutes to stop the reaction, the cells were removed by centrifugation, and the aldoses produced by HPAEC-PAD were examined. Chromatographic charts of HPAEC-PAD analysis of reaction solutions from resting cells of Pseudogluconobacter saccharoketogenes Rh47-3, Yojayella xiaoguanensis JCM30821, and Yojayella tibetensis JCM19854 are shown in Figures 3, 4, and 5, respectively. All resting cells showed similar reactivity to free Ps-ADH, producing the corresponding aldoses from their respective sugar alcohols.

[0064] Identification of D-glucose produced by oxidation reaction and separation of L-glucose We identified the D-glucose produced by the oxidation reaction of D-sorbitol using the Rh47-3 and JCM30821 resting bacterial cells mentioned above. The amount of D-glucose in the D-sorbitol reaction solution was measured using Glucose CII-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) and HPAEC-PAD. The Rh47-3 resting bacterial cell reaction solution contained 1.4 mM D-glucose as measured by HPAEC and 1.8 mM D-glucose as measured by Glucose Test. The JCM80321 solution contained 1.7 mM and 1.9 mM D-glucose, respectively. The glucose amount measured in the glucose test was quantified using the simplified calculation formula in the accompanying instructions without drawing a calibration curve. Although there was a slight difference in glucose concentration compared to the HPAEC-PAD method, almost the same concentration of D-glucose was detected in the reaction solution. Furthermore, five times the amount of glucose test enzyme solution specified in the accompanying instructions was added to the resting bacterial cell reaction solution and reacted at 37°C for 2 hours. After the reaction, the solution was boiled for 5 minutes to stop the enzymatic reaction, and then analyzed using HPAEC-PAD (Figure 6). In both Rh47-3 and JCM30821, the D-glucose peak, which was visible without enzymatic treatment (Blank), disappeared after enzymatic treatment (Glc test+), and a D-glucono-1,5-lactone peak was generated. These results confirmed the formation of D-glucose in the reaction mixture. Furthermore, L-glucose was not affected by enzymatic treatment. D-glucose and L-glucose have the same molecular weight and differ mainly only in the orientation of their hydroxyl groups, making it difficult to isolate L-glucose from a mixture of these monosaccharides. On the other hand, as shown in Figure 6, by treating D-glucose with an enzyme such as glucose dehydrogenase, which has high substrate specificity, it is possible to selectively oxidize only D-glucose. By oxidizing D-glucose to D-glucono-1,5-lactone, an acidic sugar lactone, it can be easily separated from L-glucose, a neutral sugar. Therefore, treatment with glucose dehydrogenase is an effective method for separating L-glucose. [Examples]

[0065] Identification of the structure of allose produced during alitol oxidation. To 2.5 μl of allitol oxidation reaction solution (containing 4.5 mM allose in terms of D-glucose equivalent) (final concentration of allose, 0.45 mM), potassium ferricyanide (final concentration 12.5 mM), McKiebenn buffer (1x final concentration), and Ps-ADH (final concentration 0.23 U / ml) were added to make a total volume of 25 μl, and the mixture was reacted at 30°C for 18 hours (after enzymatic treatment). Ps-ADH was not added to the control (before enzymatic treatment). In addition, for D-allose and L-allose standards, D-allose and L-allose were added to the reaction solution to achieve a final concentration of 4 mM, instead of the allitol oxidation reaction solution. After incubating the reaction solution at 30°C for 18 hours, the reaction was stopped by heating at 100°C for 5 minutes, and the mixture was analyzed using HPAEC-PAD.

[0066] In the D-allose standard, the D-allose peak disappeared after Ps-ADH treatment, but the allose peak area in the alitol oxidation reaction solution after enzymatic treatment remained almost unchanged from before treatment. Furthermore, the 8.1 min peak area, which is large during D-allose oxidation, hardly increased in the enzymatically treated solution. On the other hand, although a peak resembling an oxide was observed in the L-allose standard reaction solution, most of the L-allose remained. These results indicate that most of the allose contained in the alitol oxidation reaction solution is L-allose. However, although it was previously stated that the 8.1 min peak, which shows a characteristic increase during D-allose oxidation, hardly increased in the enzymatically treated solution, when comparing this peak to the pre-reaction (pre-enzymatic treatment) state, it increased, albeit very slightly, after enzymatic treatment. This result suggests the possibility that a very small amount of D-allose is generated (Figure 7).

[0067] In summary, the present invention is as follows: This invention relates to a technology that can more efficiently produce rare sugars, particularly aldohexoses, using inexpensive sugars as raw materials. Specifically, it involves producing a particular rare sugar using the cells of a particular microorganism or a dehydrogenase that oxidizes the sugar alcohol produced by that microorganism in the presence of a specific substrate sugar alcohol. The solution involves using D-sorbitol, L-sorbitol, L-iditol, L-talitol, and allitol as substrates and a specific Ps-ADH enzyme to produce L-growth, D-growth, L-idose, L-altrose, and L-allose.

Claims

1. A method for producing L-growth, characterized by producing it in the presence of D-sorbitol using a dehydrogenase that oxidizes the cells of microorganisms of the genera Pseudogluconobacter, Yojayella, Paradevosia, Methyloteligna, or Devosia, or the sugar alcohols of these microorganisms.

2. A method for producing D-growth, characterized by producing it using a dehydrogenase that oxidizes the microbial cells described in claim 1 or a sugar alcohol derived from the same microorganism in the presence of L-sorbitol.

3. A method for producing L-idose, characterized by producing it using a dehydrogenase that oxidizes the microbial cells described in claim 1 or a sugar alcohol derived from the same microorganism in the presence of L-iditol.

4. A method for producing L-althrose, characterized by producing it using a dehydrogenase that oxidizes the microbial cells described in claim 1 or a sugar alcohol derived from the same microorganism in the presence of L-thalitol (L-althritol).

5. A method for producing L-allose, characterized by producing it using a dehydrogenase that oxidizes the microbial cells described in claim 1 or a sugar alcohol derived from the same microorganism in the presence of allitol.

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