JP2011254780A - Sugar derivative and production method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of producing a novel sugar derivative by utilizing C6 sugars such as glucose and C5 sugars such as xylose by using microorganisms.SOLUTION: This method of producing chemical compound expressed by the following formula (1) comprises: culturing microorganisms belonging to the genus Myceligenerans in a culture medium including a utilizable carbon source; and generating the chemical compound expressed by the following formula (1) in the culture medium.

Description

  The present invention relates to a novel sugar derivative and a method for producing the same.

  In recent years, research using renewable biomass in place of fossil fuels has been underway. In particular, woody biomass, which is cellulosic biomass, does not compete with food use, and has a large accumulation amount and production amount, and is expected to promote its use as biomass. In woody biomass, the ratio of sugars (saccharified sugars) produced by saccharification (hydrolysis) treatment is about 40% for C6 sugars such as glucose and about 27% for C5 sugars such as xylose. It is characterized by a relatively high ratio. Regarding C6 sugar obtained by saccharification treatment of cellulosic biomass, various studies such as production of succinic acid using glucose as a raw material in addition to use as a raw material of bioethanol (Non-patent Document 1) have been promoted. . On the other hand, since C5 sugars such as xylose cannot be fermented with yeast or the like that is usually used for saccharification treatment, development of an effective utilization method is strongly desired.

Journal of Environmental Biotechnology, 2004, 4, 63-67

  An object of the present invention is to provide a method for producing a novel sugar derivative using a C6 sugar such as glucose or a C5 sugar such as xylose using a microorganism. Moreover, this invention makes it a subject to provide the microorganisms used for the said manufacturing method. Furthermore, an object of the present invention is to provide a novel sugar derivative.

The present inventors thought that if a novel sugar derivative can be obtained using oligosaccharides or monosaccharides obtained by saccharification treatment of cellulosic biomass as raw materials, it can lead to effective utilization of cellulosic biomass.
In view of the above problems, the present inventors have conducted intensive studies. As a result, it has been found that a bacterium belonging to the genus Myceligenerans produces a compound represented by the following formula (1) using glucose, xylose or the like as a raw material. The present invention has been completed based on this finding.

The present invention is characterized in that a microorganism belonging to the genus Myceligenerans is cultured in a medium containing an assimitable carbon source to produce a compound represented by the following formula (1) in the medium. The present invention relates to a method for producing a compound represented by the following formula (1).

The present invention also relates to a microorganism that belongs to the genus Myceligenerans and has the ability to produce the compound represented by the formula (1) from an assimitable carbon source.

  Furthermore, this invention relates to the compound represented by said Formula (1).

  According to the present invention, it is possible to provide a method for producing a novel sugar derivative using a microorganism using a C6 sugar such as glucose or a C5 sugar such as xylose. Moreover, according to this invention, the microorganisms used for the said manufacturing method can be provided. Furthermore, according to the present invention, a novel sugar derivative can be provided. The sugar derivatives obtained according to the present invention are useful as starting materials for chemical / organic synthesis.

It is the figure which detected the characteristic peak which is not in a culture medium in retention time 20.05 minutes by HPLC analysis. FIG. 1 (a) shows the HPLC analysis result of the culture supernatant cultured in the LBX medium adjusted to pH 8, and FIG. 1 (b) shows the HPLC analysis result of the culture supernatant cultured in the LBG medium adjusted to pH 6. ) Shows the results of HPLC analysis of the culture supernatant cultured in LBG medium adjusted to pH 8. It is a figure which shows that the fractionated characteristic peak is refine | purified.

Hereinafter, the production method and microorganism of the present invention will be described.
The production method of the present invention is characterized by culturing a microorganism belonging to the genus Myseligenens in a medium containing an assimitable carbon source to produce a compound represented by the following formula (1) in the medium. To do.

  The microorganism used in the production method of the present invention is not particularly limited as long as it is a microorganism belonging to the genus Myseligenans and can produce the compound represented by the formula (1).

  The production method of the present invention has a 16S rRNA gene comprising the base sequence shown in SEQ ID NO: 1 or the base sequence shown in SEQ ID NO: 1 and a base sequence equivalent in molecular phylogeny based on the base sequence of 16S rRNA gene. It is preferable to use a microorganism belonging to the genus Myceligenellan that has the ability to produce the compound represented by the formula (1) and exhibits the following mycological properties.

(Mycological properties)
(1) Gram staining positive (2) Bacterial shape Spherical (3) Motility None (4) Spore formation (5) Formation of branched hyphae Formation (6) Formation of aerial hyphae No formation

The microorganism used in the production method of the present invention is the ability to produce the compound represented by the formula (1) among the microorganisms belonging to the genus Myseligenellan having the ability to produce the compound represented by the formula (1). More preferably, it is Myceligenerans sp. KSM-TB839 strain. The Myceligenerans sp. KSM-TB839 strain was issued on September 17, 2009 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1-1 Tsukuba, Tsukuba City, Ibaraki Prefecture). Deposited at the center center 6) under the deposit number FERM P-21850.

  Microorganisms belonging to the genus Myseligenerens that can be used in the present invention can be obtained from, for example, growth-inhibiting compound-assimilating bacteria. Examples of the growth inhibitory compound include furfuryl alcohol, but are not limited thereto. These assimilation properties can be used in the present invention, for example, by culturing in Yeast Nitrogen Base (manufactured by Difco) using furfuryl alcohol as the sole carbon source and using the following mycological properties as an index. A microorganism belonging to the genus Serigenelans can be found.

The mycological properties of Myceligenerans sp. KSM-TB839 strain (Accession No. FERM P-21850) that can be particularly preferably used in the production method of the present invention are as follows.

(A) Culture properties Difco ^™ Marine Agar 2216 medium (BD): grows well at 25 ° C. for 3 days 2. Growth on Yeast Nitrogen Base (YNB) agar medium containing 0.5% furfuryl alcohol: grows well at 25 ° C. for 7 days LB agar medium (0.125% LB medium "Digo" (Nippon Pharmaceutical Co., Ltd.), 0.9% artificial seawater, 1.5% agar, pH 7 growth: grows well at 25 ° C for 7 days

(B) Morphological properties The colony morphology after culture for 72 hours at 25 ° C. in Difco ^™ Marine Agar 2216 medium (manufactured by BD) is shown.
1. Diameter: 1.0-2.0mm
2. 2. Color tone: Yellow Shape: Circular 4. Raised state: lenticular Perimeter: thread-like 6. 6. Surface shape: smooth Transparency: opaque 8 Viscosity: adheres to the medium

(C) Physiological properties Growth temperature test 45 ° C Grows 50 ° C Does not grow 2. 2. Catalase reaction: positive Oxidase reaction: negative 4. 4. Anaerobic growth: negative Acid / gas production from glucose Acid production: negative Gas production: negative O / F test using MOF medium Oxidation: negative Fermentation: negative Oxidation test using API150CHE Glycerol: negative Erythritol: negative D-arabinose: negative L-arabinose: positive ribose: negative D-xylose: positive L-xylose: negative adonitol: negative β-methyl-xyloside: positive galactose: negative glucose: Positive Fructose: Negative Mannose: Positive Sorbose: Negative Rhamnose: Negative Dulcitol: Negative Inositol: Negative Mannitol: Negative Sorbitol: Negative α-Methyl-D-Mannoside: Negative α-Methyl-D-glucoside: Positive N-Acetylglucosamine: Negative Amygdalin : Negative Arbutin: Positive Esculin: Positive Salicin: Negative Cellobiose: Positive Maltose: Negative Lactose: Negative Melibiose: Negative Sucrose: Sex Trehalose: Positive Inulin: Negative Meretitose: Negative Raffinose: Negative Starch: Negative Glycogen: Negative Xylitol: Negative Gentiobiose: Positive D-Tulanose: Positive D-Lixose: Positive D-Tagatose: Negative L-Fucose L D-arabitol: negative L-arabitol: negative gluconate: negative 2-ketogluconate: negative 5-ketogluconate: positive Optimal growth temperature range: 20-30 ° C
9. Optimal growth pH range: 6-8
10. Maximum growth temperature: 45 ° C
11. Growth possible pH range: 4-13

(D) Chemical taxonomic properties The nucleotide sequence encoding 16S rRNA of Myceligenerans sp. KSM-TB839 strain is the Myceligenerans xiligouense strain (XLG9A10.2). It has 98.9% identity with 16S rRNA and 95.2% with 16S rRNA of the Isoptericola dokdonensis strain (DQ387860).

(E) Other characteristics The above-mentioned properties support that Myceligenerans sp. KSM-TB839 is a genus of Myceligenans, but are completely consistent with these properties. Since the bacterium species to be found is not found in the genus Myseligenanes, it is identified as Myceligenanes sp.

  In the production method of the present invention, the 16S rRNA gene belongs to the genus Myseligenanes, and the 16S rRNA gene is based on the base sequence shown in SEQ ID NO: 1 and the base sequence of 16S rRNA possessed by the 16S rRNA of the Myseligenance sp. A microorganism having an equivalent base sequence in molecular phylogeny and exhibiting the above bacteriological properties and capable of producing the compound represented by the formula (1) from an assimitable carbon source can be preferably used. .

Methods for studying the evolution of organisms and genes based on molecular phylogenetic trees have been established as molecular phylogeny (see, for example, Shigeo Kimura, Introduction to Molecular Evolution Chemistry (Baifukan), pages 164-184, “Seven Molecular Phylogenetic Trees”). "How to make and evaluation"). The molecular phylogenetic tree based on the base sequence of the 16S rRNA gene is based on the base sequence of the 16S rRNA gene of a known microorganism that is presumed to be the same or similar to the microbe from the mycological properties of the base sequence of the 16S rRNA gene of the target microorganism. At the same time, it can be obtained by calculating multiple alignments and evolutionary distances and creating a phylogenetic tree based on the obtained values. The base sequence of a known microorganism 16S rRNA gene used for the creation of a molecular phylogenetic tree can also be obtained by an identity search of an existing database. Here, the evolution distance refers to the total number of mutations per locus (sequence length) between certain genes.
In the present invention, “equivalent in molecular phylogeny” refers to a microorganism recognized as the same genera in the molecular phylogenetic tree, and among them, if the identity of the base sequence of 16S rRNA gene is 97% or more, It is more closely related, and if it is 99% or more, it is recognized that there is a high possibility of being the same species. Here, the identity of the base sequences refers to the identity (%) of the maximum base sequences obtained by aligning the two base sequences to be compared by introducing a gap as necessary. Alignment for the purpose of determining the identity of the base sequence can be performed using a conventional method. For example, publicly available computer software such as BLAST, DNASIS Pro (Hitachi Software Engineering Co., Ltd.) ) And commercially available software such as GENETYX (Genetics Co., Ltd.) can also be used.

  A compound represented by the above formula (1) is cultured in a medium containing an assimitable carbon source under aerobic conditions by culturing a microorganism belonging to the genus Myseligenans as described above in the culture medium. Can be generated. Microorganisms belonging to the genus Myseligenans can be used alone, but any one type or two or more types of microorganisms may be used simultaneously.

  In the present invention, a liquid medium is usually used as the medium. Specific examples of such a medium include Luria-Bertani medium. The carbon source that can be assimilated is not particularly limited as long as it produces the compound represented by the above formula (1) when a microorganism belonging to the genus Myseligenus is aerobically cultured. Examples thereof include glucose, arabinose and sucrose, and xylose is preferable. An assimitable carbon source can be obtained by purchasing a commercial product or synthesizing it by a method well known to those skilled in the art.

  In the production method of the present invention, saccharified sugars such as oligosaccharides and monosaccharides produced by saccharification of biomass can also be used as a carbon source. The saccharified saccharide of biomass can be used as it is as an assimitable carbon source, or may be separated and purified by methods well known to those skilled in the art. In recent years, the use of biomass has been increasing, and it is predicted that saccharified sugar will continue to be produced. If such saccharified sugar can be effectively used as a raw material, it has an important meaning in terms of environment and industry.

  The concentration of the assimilable carbon source contained in the medium is not particularly limited as long as the microorganism belonging to the genus Myceligenera can produce the compound represented by the formula (1). It is desirable to use in the range of 10 g / dl. An assimitable carbon source can be added to the medium stepwise.

  Furthermore, a nitrogen source, inorganic salts, various organic substances, inorganic substances, surfactants or commonly used antifoaming agents, etc., necessary for the growth of microorganisms belonging to the genus Mycelenelans used in the present invention may be added as necessary. Can do. These medium components can also be added to the medium as necessary.

  Inoculation of a microorganism belonging to the genus Myseligenerus into, for example, a method of directly ingesting a microorganism belonging to the genus Myseligenus suspended in physiological saline or the like into the production medium, Can be performed by directly inoculating one platinum ear with a microorganism belonging to the above.

  The culture temperature of the main culture is within a range in which a microorganism belonging to the genus Mycelenelans can grow, that is, usually 20 to 45 ° C, preferably 20 to 30 ° C. Further, the pH of the medium is usually adjusted to 6 to 8, preferably in the vicinity of 7. The culture period varies depending on the type of medium used and the concentration of the carbon source, and is usually about 24 hours to 14 days. The culture in the present invention may be terminated when the nutrient source of the medium is utilized to the maximum and the accumulated amount of the compound represented by the formula (1) produced in the culture solution reaches the maximum. preferable.

  In addition, the kind and production amount of the compound represented by the formula (1) in the culture solution can be measured using a usual method such as high performance liquid chromatography or LC-MS. The compound represented by the formula (1) obtained by the present invention is a well-known means usually used in the art, for example, filtration, centrifugation, vacuum concentration, ion exchange or adsorption chromatography, solvent extraction, distillation, It can be collected from the culture solution by using an appropriate combination of operations such as crystallization as necessary, but is not particularly limited to these methods.

As described above, the compound represented by the formula (1) can be produced by using the production method and microorganism of the present invention. The compound represented by the formula (1) is glucose 2,6 succinate, which is a novel sugar derivative.
In the glucose 2,6 succinate represented by the formula (1), since the hydroxyl groups at the 2nd and 6th positions of the glucose skeleton are masked, only the hydroxyl group at the 1st position of the glucose skeleton can be reacted specifically. . In general, in a chemical synthesis reaction, in order for a reaction to proceed at a hydroxyl group at a desired position of a sugar molecule, it is necessary to mask the hydroxyl group at another position by some method so that the reaction cannot be performed. Such a masking operation is important for improving selectivity in a chemical reaction. For example, although it is known that the reactivity of hydroxyl groups in sugars decreases in the order of 1st, 6th, 2nd, 3rd, 4th (Non-Patent Document 2), only the 1st position is specific. In order to cause the glucosylation reaction, it is necessary to mask at least the 6th and 2nd positions. In other words, if the highly reactive 6th and 2nd positions are masked in advance after the 1st position, it is easy to react only the 1st position without masking operation. Therefore, the compound represented by the formula (1) is useful as a starting material in the field of chemical synthesis represented by the fields of medicine, cosmetics and food.

  Moreover, the compound represented by the formula (1) is a novel compound that has not been reported so far, and the functionality of the molecule itself is also expected. For example, taking an analog of glucose as an example, [1 gF] -fluoro-2-deoxy-D-glucose is used for PET diagnosis of oral cancer, 3-methylglucose is used for evaluating sugar uptake activity, and 2-deoxyglucose is used. For mutant breeding of yeast, α-methyl-D-glucopyranoside has been used to study vesicular glucose transport in the rabbit kidney. Therefore, such a wide range of uses can be expected for the compound represented by the formula (1) of the present invention.

EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited to this.
Test example (1) Acquisition of strain 0.85% (w / v) A suitable amount of soil (seabed soil in Sakata Port, Chiba Prefecture) was added to 5 mL of saline, stirred, allowed to stand, and then adjusted to pH 7 0.67% (W / v) YNB (manufactured by Difco), 1.8% (w / v) Daigo artificial seawater (Nippon Pharmaceutical), 0.5% (v / v) furfuryl alcohol (Wako Pure Chemical Industries), 1.5 An appropriate amount was smeared on an agar medium composed of% (w / v) Bacto Ager (Wako Pure Chemical Industries) and cultured at 25 ° C. for 30 days. The grown strain was monocolonized to obtain KSM-TB839 strain.

(2) Identification morphological observation and BARROW et al. Method strain (GIBARROW and RKAFELTHAM: Cowan and Steel 's Manual for the Identification of Medical Bacteria, 3 rd edition, 1993, Cambridge University Press reference) to Based catalase reaction, the oxidase reaction test conducted It was. As a result, the KSM-TB839 strain is a gram-positive filamentous gonococcus having no motility (mycelial width: 0.7-0.8 μm), forms short-chain spores, does not grow under anaerobic conditions, and is at 45 ° C. It grew but did not grow at 50 ° C., formed yellow colonies on MB2216 agar medium, did not form aerial mycelium, showed positive for catalase reaction, and negative for oxidase reaction.

  When API150CHE (bioMerieux, Lyon, France) was used for the oxidation test and the like, erythritol, D-arabinose, ribose and the like were not oxidized, and L-arabinose, β-methyl-D-xyloside, glucose and the like were not oxidized. Oxidizing properties were shown.

  From the results of physiological and biochemical tests, the KSM-TB839 strain has similarities to the properties of Myseligenance xyligoens, but β-methyl-D-xyloside and α-methyl-D-glucoside are oxidized. , Rhamnose, mannitol, salicin, etc. are different in that they do not oxidize, supporting that they are bacteria of the genus Myceligenerens, but no consistent genus species were found.

  PCR was carried out according to a conventional method using the chromosomal DNA of the KSM-TB839 strain as a template and the oligonucleotide consisting of the base sequence of SEQ ID NO: 2 and the oligonucleotide consisting of the base sequence of SEQ ID NO: 3 as primers (Nakakawa Atsuyoshi, Kawasaki) Hiroko: Gene analysis method 16S rRNA gene sequencing method, edited by the Japanese Society for Actinomycetes Classification and identification of actinomycetes 88-117pp, The Chemical Society of Japan Office, 2001), a region encoding 16S rRNA was amplified. The base sequence of the obtained amplified fragment was decoded, and a partial sequence of 16S rRNA (SEQ ID NO: 1: 1484 bases) was determined.

98.9% between the 16S rRNA of My Seri Gene Lance Kishirigoensu (Myceligenerans xiligouense) Co., Ltd. (XLG9A10.2), 95.2% between the 16S rRNA of Isoputerikora-Dokudonenshisu (Isoptericola dokdonensis) Co., Ltd. (DQ387860) , Had the same identity. Therefore, the possibility of belonging to the KSM-TB839 strain and Myseligenens xyligoens is also conceivable, but since a difference of 14 bases is observed in each case, the possibility of difference cannot be denied.

  From the above results, it was identified that the KSM-TB839 strain was Myceligenene sp.

Examples Production of sugar derivatives Search for xylose converting substance and glucose converting substance LBX medium (0.25% LB medium “Digo” (Nippon Pharmaceutical), 0.9% artificial seawater, 0.5% xylose) adjusted to pH 6 or pH 8 or pH 6 or pH 8 Inoculated with Myeligenellans sp. KSM-TB839 in 1 platinum ear in LBG medium (0.25% LB medium “Digo” (Nippon Pharmaceutical), 0.9% artificial seawater, 0.5% glucose) And cultured for 7 days with shaking (30 ° C., 250 rpm). The culture solution was centrifuged (12000 rpm, 20 minutes), and the supernatant fraction was fractionated.

Analysis of the compounds contained in the culture supernatant was performed using a CAPCEL PAK C18 AQ (SHISEIDO) with a Hitachi LaChrom Elite apparatus (HITACHI). The sample is appropriately diluted with (0.1% (v / v) formic acid solution), and the flow rate is 0.5 mL / min, the flow rate is 0.5 mL / min, the column temperature is 30 ° C., the sample injection amount is 10 μL, and 0.2 μm. HPLC was performed under a concentration gradient condition (Table 1) from a 1% formic acid solution to a 0.1% formic acid solution containing 30% acetonitrile, and a Corona ^™ CAD ^™ charged particle detector (ESA) and a UV detector (HITACHI) Analysis). All analysis samples were used after being filtered through DISMIC-13CP Cellulose Acetate 0.2 μm (manufactured by ADVANTEC).

  As a result, in the culture supernatant of the LBX medium adjusted to pH 8 and the LBG medium adjusted to pH 6 and pH 8, a characteristic peak (elution time 20.05 minutes) of a substance not present in the medium not cultured was observed. (FIG. 1).

2. Purification of characteristic peak components Myceligenanes sp. KSM- in LBX medium (0.25% LB medium “Digo” (Nippon Pharmaceutical), 0.9% artificial seawater, 0.5% xylose) adjusted to pH 8) TB839 strain was inoculated with 1 platinum ear and cultured with shaking (30 ° C., 250 rpm) for 7 days. The culture solution was centrifuged (12000 rpm, 20 minutes), and the supernatant fraction was fractionated. The fractionated supernatant was filtered with DISMIC-13CP Cellulose Acetate 0.2 μm (ADVANTEC).

The fractionated supernatant was concentrated as appropriate, and using an Inertsil ODS3 preparative column (GL Science) stabilized with 30% methanol as the moving bed, flow rate 7.5 mL / min, column temperature 40 ° C., sample injection volume 200 μL Then, HPLC was performed under conditions of an analysis time of 20 min, and analysis was performed with a UV (210 nm) detector, and characteristic peaks were collected. The fraction collected was subjected to HPLC analysis according to the conditions of Example 1, and it was confirmed that a characteristic peak was purified (FIG. 2). It was found that the productivity was 0.6 g / L from the weight of the compound having a characteristic peak separated.

3. Identification of characteristic peak components Characteristic peaks were analyzed by NMR. In the analysis by NMR, a dried sample (4 mg) was dissolved in 500 μL of a solvent (mixing ratio, D2O: CD3OD = 1: 1), and 1H NMR (600 MHz) and 13C NMR (150 MHz) were AV-600 (BRUKER). ). The results are shown in Table 2.

  When the measurement result was analyzed, it was found that this compound was glucose 2,6 succinate represented by the following formula (1).

When the melting point of this compound was measured using a melting point analyzer MPA-50 type (Miyamoto Riken Kogyo Co., Ltd.), it was found that the melting point was 303.0 to 305.0 (decomposition temperature).

  From these results, it was found that the compound represented by the formula (1) can be produced by the production method of the present invention or the microorganism of the present invention using a medium containing xylose, glucose and the like.

Claims (9)

A microorganism belonging to the genus Myceligenerans is cultured in a medium containing an assimitable carbon source to produce a compound represented by the following formula (1) in the medium: The manufacturing method of the compound represented by (1).
  The production method according to claim 1, wherein the assimilable carbon source is xylose or glucose. The microorganism belonging to the genus Myceligenerans comprises the base sequence shown in SEQ ID NO: 1 or the base sequence shown in SEQ ID NO: 1 and the base sequence equivalent in molecular phylogeny based on the base sequence of the 16S rRNA gene The microorganism according to claim 1 or 2, which has a 16S rRNA gene, exhibits the following mycological properties, and has the ability to produce a compound represented by the following formula (1) from an assimitable carbon source. Production method.
(1) Gram staining positive (2) Bacterial shape Spherical (3) Motility None (4) Spore formation (5) Formation of branched hyphae Formation (6) Formation of aerial hyphae No formation
The method according to any one of claims 1 to 3, wherein the microorganism belonging to the genus Myceligenerans is Myceligenerans sp. KSM-TB839 strain (FERM P-21850). A microorganism belonging to the genus Myceligenerans and capable of producing a compound represented by the following formula (1) from an assimitable carbon source.
  6. The microorganism according to claim 5, wherein the assimilable carbon source is xylose or glucose. It has a 16 SrRNA gene comprising the base sequence shown in SEQ ID NO: 1, or a base sequence shown in SEQ ID NO: 1 and a molecular phylogenetic equivalent base sequence based on the base sequence of 16S rRNA gene, and has the following mycological properties The microorganism according to claim 5 or 6, wherein
(1) Gram staining positive (2) Bacterial shape Spherical (3) Motility None (4) Spore formation (5) Formation of branched hyphae Formation (6) Formation of aerial hyphae No formation Myceligenerans sp. KSM-TB839 strain (FERM P-21850). A compound represented by the following formula (1).