KR20000011556A - Novel microorganisms and method for producing xylitol or D-xylulose - Google Patents

Abstract

PURPOSE: Xylitol and D-xylullose are prepared by a microbe from glucose. CONSTITUTION: Osmophillic microorganism collected from earth is cultured at 30 degree C for 5 days in a medium containing 20 %(w/v) D-glucose, 0.1 % urea and 0.5- 5 yeast extracts to give the microorganism to produce xylitol and D-xylullose. A medium containing 0.5- 5 yeast extracts and 0.1- 5 urea is sterilized at 120 degree C for 20 minutes. A strilized glucose is added to the medium to contain 20 % (w/v) of glucose, followed by culturing at 30 degree C for 5 days to give the xylitol and D-xylullose.

Description

Novel microorganisms and method for producing xylitol or D-xylulose

The present invention utilizes novel microorganisms having the ability to produce xylitol or D-xylulose and microorganisms having the ability to produce xylitol or D-xylose using xylitol or D-xylose. It is about how to produce. D-xylulose is useful as a material for xylitol production, and xylitol is useful as a sweetening agent in fields such as the food industry.

The demand for xylitol, a naturally occurring sugar alcohol, is expected to increase in the future. Xylitol is a promising low-calorie sweetener because it is low in calories and displays a comparable sweetness compared to sucrose. In addition, due to its anti-caries properties, it is used as a sweetener for preventing caries. In addition, xylitol does not elevate glucose concentration, and thus is used for latex therapy in the treatment of diabetes. For this reason, the demand for xylitol is expected to increase in the future.

The current industrial production of xylitol is primarily in the hydrogenation of D-xylose as described in US Pat. No. 4,008,285. D-xylose, used as crude, is obtained by hydrolyzing plant material such as wood, straw, corn cobs, oat husks and other xylan-rich substances.

However, D-xylose produced by hydrolysis of plant material has the disadvantage that it is rather expensive and high production cost. For example, low yields of hydrolysis treatment of plant materials result in low purity of the produced D-xylitol. Therefore, the acid used in the hydrolysis and dye must be removed by the ion exchange treatment after the hydrolysis treatment, and the D-xylose obtained must also be crystallized to remove other semicellulosic saccharides. In order to obtain D-xylose suitable for food, further purification is required. These ion exchange treatments and crystallization treatments lead to an increase in production costs.

Thus, several methods have been developed for producing xylitol, which are not wasted at all using readily available crude materials. For example, a method of producing xylitol using another pentitol as starting material has been developed. One such readily available pentitol is D-arabitol, which can be produced by using yeast (Can J. Microbiol., 31, 1985, 467-471; J. Gen Microbiol., 139, 1993, 1047-54). As a method for producing xylitol by using D-arabitol as a crude substance, a method reported in Applied Microbiology., 18, 1969, 1031-1035 can be mentioned, which method is called debariomy After producing D-arabitol from glucose by fermentation with Debaryomyces hansenii ATCC20121, D-arabitol is converted to D-xylulose using Acetobacter suboxydance, Converting D-xylulose to xylitol by the action of the Candida guilliermondii var. Soya.

EP 403 392A and EP 421 882A produce D-arabitol by fermentation with osmosis-resistant yeast and then convert D-arabitol into the genus Acetobacter, Glucobacter. Bacteria belonging to the genus Gluconobacter or Klebsiella are used to convert D-arabitol to D-xylulose and by the action of glucose (xylose) isomerase from D-xylulose. A method comprising forming a mixture of xylose and D-xylulose and converting the obtained mixture of xylose and D-xylose to xylitol by hydrogenation is described. It also describes the production of xylitol, which comprises preconcentrating xylose and converting xylose to xylitol by hydrolysis in a mixture of xylose and D-xylulose.

However, the above-mentioned production method of xylitol uses D-arabitol produced by fermentation as starting material, which is converted by a number of method steps. Therefore, these methods are complex and unsatisfactory in terms of process economy compared to extraction-based methods.

Therefore, there is a need for microorganisms having the ability to produce xylitol or D-xylose from single glucose by fermentation starting from glucose as used for the production of other saccharides and sugar alcohols. However, such bacteria with the ability to produce xylitol or D-xylulose have not been reported yet.

On the other hand, breeding of xylitol fermented bacteria has been attempted by using gene amplification techniques. International Publication No. WO94 / 10325 aravitol ferments arabitol dehydrogenase gene originating from bacteria belonging to the genus Krebsela and xylitol dehydrogenase gene originating from bacteria belonging to the genus Pichia. It describes the production of xylitol from glucose by fermentation using recombinant microorganisms obtained by introduction into microorganisms (Yeast belonging to the genus Candida, Torulopsis or Zygosaccharomyces). . However, production of 15 g / L xylitol from 400 g / L glucose, although reported for the recombinant microorganism, does not actually reach useful levels of accumulation. Moreover, since the recombinant microorganisms described above are introduced using genes originating from different species, information on their stability may not be considered sufficient.

The present invention has been accomplished in the aforementioned aspect of the art, and an object of the present invention is to provide a microorganism having the ability to produce xylitol or D-xylose from glucose by fermentation and xylitol or It is to provide a method for producing D-xylulose.

In order to achieve the above object, the present inventors investigated microorganisms having the ability to produce xylitol or D-xylose from glucose by fermentation. For direct production of sugar alcohols by fermentation of microorganisms such as yeast, the production of glycerol by using Zygosaccharomyces acidifaciens in addition to the above-mentioned aviitol fermentation is described in Arch. Biochem., 7, 257-271 (1945)], Production of erythrodotol by using yeast belonging to the genus Trychosporonoides [Trychosporonoides sp., See Biotechnology Letters, 15, 240-246 (1964) ] Is reported. All of these yeasts with sugar alcohol production ability exhibit good osmotic pressure, ie, the ability to grow well in culture media with high osmotic pressure. Thus, although microorganisms with xylitol production capacity have not been found in the osmotic yeast, the inventors have considered that new microorganisms with xylitol production capacity may be present in the osmotic microbes. Was screened intensively. As a result, the inventors have discovered microorganisms having the ability to produce xylitol and D-xylulose from glucose among the osmotic microorganisms. These microorganisms were evaluated to be novel bacteria in terms of phylogeny, based on the nucleotide sequence of the 16S rRNA gene. The present invention has been accomplished based on this finding.

Therefore, the present invention

A 16S rRNA comprising a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 1 in terms of molecular systematics based on the nucleotide sequence of SEQ ID NO: 1 or the 16S rRNA sequence to produce xylitol or D-xylose from glucose Capable microorganisms belonging to the Actetobacteracea family, and

Producing a xylitol or D-xylulose from glucose with a 16S rRNA gene comprising the nucleotide sequence of SEQ ID NO: 2 homologous to the nucleotide sequence in molecular phylogeny on the basis of the nucleotide sequence of SEQ ID NO: 2 or the 16S rRNA sequence Provide microorganisms with the capacity.

Examples of the above-mentioned microorganisms include, for example, microorganisms belonging to the genus Asia or Zucharibacter, more particularly Asia ethanolifaciens or Zucharibacter floricola. Includes strains. Asia ethanollipciens is a novel species (sp.nov.) Previously named by the inventors. Zucarybacter genus and Zucarybacter floricola, respectively, are genus (nov.) And novel species previously named by the inventors.

Specific examples of the above-mentioned microorganisms include, for example, strain P528 (FERM BP-6751), strain S877 (FERM BP-6752), strain S1009 (FERM BP-6753), strain S1019 (FERM BP-6754) and strain S1023 ( FERM BP-6755).

The 16S rRNA gene of strain P528 comprises the nucleotide sequence of SEQ ID NO: 1 and the 16S rRNA gene of strain S877 comprises the nucleotide sequence of SEQ ID NO: 2. The partial sequence of the 16S rRNA gene of strains S1009, S1019 and S1023 comprises the nucleotide sequence of SEQ ID NO: 3 to SEQ ID NO: 5, respectively. These nucleotide sequences are homologous to the nucleotide sequence of SEQ ID NO: 2 in terms of molecular systematics based on the nucleotide sequence of the 16S rRNA.

The invention also provides for culturing a microorganism having the ability to produce xylitol or D-xylose from glucose in a medium suitable for accumulating xylitol or D-xylose in the medium and from the medium. Provided is a method for producing xylitol or D-xylose, comprising collecting xylitol or D-xylulose.

Examples of microorganisms used in the method are, for example, from 16 glucose having a 16S rRNA gene comprising a nucleotide sequence homologous to a nucleotide sequence in molecular systematics based on the nucleotide sequence of SEQ ID NO: 1 or the 16S rRNA gene sequence. Microorganisms belonging to the genus Acetobacteraceae, having the ability to produce xylitol or D-xylulose, and nucleotides homologous to the nucleotide sequence in molecular phylogenetics on the basis of the nucleotide sequence of SEQ ID NO: 2 or the 16S rRNA gene sequence Microorganisms belonging to the genus Acetobacteraceae that have the 16S rRNA gene comprising the sequence and have the ability to produce xylitol or D-xylose from glucose.

Specific examples of the above-mentioned microorganisms include, for example, microorganisms belonging to the genus Asia or Zucarybacterus, more particularly asia ethanollipassien or Zucarybacter floricola strain. Specific examples of the aforementioned microorganisms include, for example, strains P528, S877, S1009, S1019 and S1023.

The present invention also provides a method of producing ethanol, comprising culturing microbial strain P528 (FERM BP-6751) in a medium suitable for accumulating ethanol in the medium and collecting ethanol from the medium.

According to the present invention, xylitol or D-xylose can be produced efficiently from inexpensive materials such as glucose.

Ethanol can also be produced by using strain P528.

1 depicts similar bacteria based on the molecular phylogenetic tree of the microorganisms of the present invention and the nucleotide sequence of 16S rRNA.

Figure 2 shows the alignment of the partial sequence of the 16S rRNA of the xylitol producing microorganism, and compares the nucleotide sequence of SEQ ID NO: 1 to 691 nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 and SEQ ID NO: 3 to 5. The dot (·) represents a general nucleotide.

3 is a continuation of FIG. 2.

4 is a graph showing the effect of NaCl addition on the growth of microorganisms of the present invention.

5 is a graph showing the production of acetic acid when strains P528 and S877 are cultured in ethanol-added medium.

FIG. 6 is a graph showing the consumption or production of ethanol when strains P528 and S877 are cultured in ethanol-added medium.

7 is a graph showing ethanol production by strain P528.

The invention will now be described in detail later.

<1> microorganism of the present invention

The inventors have discovered novel microorganisms with the ability to produce xylitol or D-xylulose from glucose as a result of intensive screening of the osmotic microorganisms as described in the examples mentioned later. These microorganisms were named strains P528, S877, S1009, S1019 and S1023.

The microbiological properties of these strains will be mentioned below.

[1] morphological and cultural properties

The above-mentioned strains were kept at 30 ° C. for 3 days in YM medium (1% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, pH 6.0) supplemented with 11% (w / v) D-glucose. After incubation, observe with a microscope. The results are shown in Table 1.

Strain P528 S877 S1009 S1019 S1023 Cell size 0.8-1 μm x 4.5-5 μm 0.8-1 μm x 2.5-3 μm 0.8-1 μm x 2.5-3 μm 0.8-1 μm x 2.5-4 μm 0.8-1 μm x 2-2.5 μm shape Stick Stick Stick Stick Stick motility none none none none none Spores none none none none none

[2] culture characteristics

(1) agar plate culture

The strains were incubated at 30 ° C. for 3 days on YM culture plates supplemented with 11% (w / v) D-glucose and the properties observed are shown in Table 2.

Strain P528 S877 S1009 S1019 S1023 growth Great Great Great Great Great Colony Round, smooth all over the terminal Round, smooth all over the terminal Round, smooth all over the terminal Round, smooth all over the terminal Round, smooth all over the terminal surface Slightly bent Slightly bent Slightly bent curve Slightly bent Polish Geological gloss Geological gloss Geological gloss Geological gloss Geological gloss color Lemon yellow Slightly yellow Slightly yellow Slightly yellow Slightly yellow

(2) broth culture

The strains were incubated at 30 ° C. for 3 days in YM culture broth supplemented with 11% (w / v) D-glucose and the properties observed are shown in Table 3.

Strain P528 S877 S1009 S1019 S1023 Surface growth none none none none none Turbidity Very cloudy Very cloudy Very cloudy Very cloudy Very cloudy Sedimentation Massive precipitation Massive precipitation Massive precipitation Massive precipitation Massive precipitation

[3] physical properties

(1) Table 4 shows the test results for the various physical properties.

Strain P528 S877 S1009 S1019 S1023 Gram strain voice voice voice voice voice Indole production none none none none none Hydrogen Disulfide Production none none none none none Oxidase none none none none none Catalase production production production production production O-F test positivity voice voice voice voice

(2) optimum growth conditions

The optimal growth temperature and optimal pH are shown in Table 5 when the strain is incubated in YM medium supplemented with 11% (w / v) D-glucose.

Strain P528 S877 S1009 S1019 S1023 Optimum growth temperature 30 ℃ 27 ℃ 27 ℃ 27 ℃ 27 ℃ Optimal growth pH 5.0-7.0 5.0-7.0 5.0-7.0 5.0-7.0 5.0-7.0

(3) growth conditions

Table 6 shows the conditions under which growth is allowed when the strain is incubated in YM medium supplemented with 11% (w / v) D-glucose.

Strain P528 S877 S1009 S1019 S1023 Growth temperature 10-37 ℃ 10-37 ℃ 10-32 ℃ 10-32 ℃ 10-32 ℃ Growth pH 2.5-9.0 2.5-9.0 2.5-9.0 2.5-9.0 2.5-9.0

(4) The optimum sucrose concentration is shown in Table 7 when the strain is cultured in YM medium supplemented with sucrose.

Strain P528 S877 S1009 S1019 S1023 Optimum sucrose concentration 20% 10% 10% 10% 10%

(5) Strains are incubated for 5 days at 30 ° C. in a medium containing 20% (w / v) D-glucose, 0.1% urea and 0.5% yeast extract. The saccharides detected in the medium after incubation are shown in Table 8.

Strain P528 S877 S1009 S1019 S1023 Metabolites from Glucose Xylitol, D-Xylose, D-Arabitol, Sorbitol Xylitol, D-Xylose, D-Arabitol Xylitol, D-Xylose, D-Arabitol Xylitol, D-Xylose, D-Arabitol Xylitol, D-Xylose, D-Arabitol

Of the aforementioned strains, the four strains S877, S1009, S1019 and S1023 show absolute osmotic pressure, ie they can only grow in medium with high concentrations of saccharides.

The main characteristic of these five microbial strains is their ability to produce xylitol or D-xylose from glucose. Since no microorganisms producing xylitol or D-xylose from glucose have been reported at all so far, strains with microbial properties as mentioned above have been determined to be novel microorganisms.

[4] molecular systematic analysis

To determine the phylogenetic positions of strains P528, S877, S1009, S1019 and S1023, the nucleotide sequences of the 16S rRNA genes of these strains were measured and the molecules using these nucleotide sequences together with the nucleotide sequences of the 16S rRNA genes of the closest microorganism A phylogenetic tree is prepared (see FIG. 1). As a result, it was suggested that strain P528 could be a new species belonging to the genus Acetobacteraceae and a novel genus similar to the genus Acetobacter. On the other hand, strain S877 has been suggested to be a microorganism belonging to the genus Acetobacteraceae and belonging to the genus Acetobacter or new genus similar to the genus Gluconobacter. Three strains S1009, S1019 and S1023 are considered to be the same species as strain S877.

Methods for evaluating genes or organisms based on molecular phylogenetic systems have been established as molecular phylogeny [Bunshi Shinka-gaku Nyumon (Introduction of Evolutionary Molecular Biology), Section 7, Method for Preparation of Molecular Phylogenetic Tree and Evaluation about, Ed. by T. Kimura, Baifukan, Japan, pp. 164-184].

Molecular genealogy based on the nucleotide sequence of the 16S rRNA gene is the same species or similar species as the microorganism of interest, with data obtained through multiple sequence alignments and measurement of the assessment distance using the nucleotides of the 16S rRNA gene of the microorganism of interest. It can be obtained by drawing up a phylogenetic tree based on data obtained through known microbial data that are estimated to be. Nucleotide sequences of known microbial 16S rRNA genes used in the preparation of molecular phylogenetic tree can be obtained, for example, by examining useful databases based on homology. As used herein, the term "evaluation distance" means the total number of mutations per genetic position (sequence length) for a particular gene.

Numerous sequence alignments and assessment distance measurements are described, for example, in the commercial software "Phylogeny Programs, such as CLUSTAL W included in phylogenetic programs" (http://evolution.genetics.washington.edu/phylip/ commercially available in software.html) (See Thompson, DJ, et al., Nucleic Acids Res., 22, 4673-4680 (1994)). Phylogenetic genealogy can be prepared by commonly available software (e.g., Tree View, Tree drawing software for Apple Machintosh: by Roderic D., Page 1995, Institute of Biomedical and Life Sciences, University of Glasgow, UK). . Specifically, the results obtained by the operation in CLUSTAL W can be output as PHLYP format data, which can be produced in Tree View. PHLYP [Felsenstein J. (1995) Phylogenetic inference package, version 3.5.7., Department of Genetics, University of Washington, Seatle WA, USA] is also included in the phylogenetic program described above.

[5] other biochemical and physical properties

(1) Quinone type and GC content of DNA

The quinone type is ubiquinone-10 for all strains P528, S877, S1009, S1019 and S1023 and the GC content of DNA is 56.5%, 52.3%, 52.3%, 51.9% and 52.9%, respectively.

(2) acid production

Acid production from various carbon sources by the strains is shown in Table 11.

(3) Effect of NaCl Addition on Growth

Growth of strains cultured in media containing various concentrations of NaCl is shown in Table 4. Strain P528 is resistant to NaCl up to at least 2%.

(4) consumption of acetic acid and lactic acid

When the strain is cultured in a medium containing glucose as a carbon source and supplemented with acetic acid or lactic acid, all strains show lactic acid degrading ability but this ability is weak or substantially incapable of acetic acid degrading ability.

(5) Effect of Addition of Acetic Acid or Ethanol on Growth

All of the strains grow actively in medium with up to 1% acetic acid or up to 3% ethanol. All of the strains do not grow in medium to which at least 4% acetic acid or at least 5% ethanol is added.

(6) production of acetic acid and consumption of ethanol

When cultured in a medium containing glucose as the carbon source, the strain exhibits weak acetic acid productivity. This strain does not show significant ethanol consumption and strain P528 shows ethanol production.

[6] Phenotypic comparison of microorganisms of the invention with other acetic acid bacteria

Strains P528, S877, S1009, S1019 and S1023, and known acetic acid bacteria, Asia bogorensis, Acetobacter aceti, Gluconobacter oxydans, Glue By Gluconacetobacter liquefaciens and Acidomons methanolica (The Congress of the Japan Society for Bioscience, Biotechnology, and Agrochemistry, 1999, Lecture Abstracts, p. And p. 66). The phenotypic comparison results are shown in Table 9. Asia bogorensis is a microorganism belonging to the genus (nov. Nov.) And new species (sp. Nov.) Reported at a conference held by Yamada et al. For strains P528, S877, S1009, S1019 and S1023, acetic acid production, ethanol production, DNA nucleotide composition and main quinones are measured as described in Examples 5 and 6. Other properties are described by Asai et al. [Asai, T. et al., J. Gen. Appl. Microbiol., 10 (2), p. 95, 1964].

As shown in Table 9, strain P528 is similar to asia borosiene but, although this strain is weak, it produces acetic acid from ethanol and the GC content of the nucleotide composition of DNA (59 to 59). 61), which is significantly lower than Asia bogorensis in that it is 56.5. Strains S877, S1009 and S1023 differ from other acetic acid bacteria in that their acetic acid production from ethanol is weak, they can grow in the presence of 30% glucose, and do not exhibit acid production from ethanol.

Based on the above results, strain P528 was identified as a new species belonging to the genus Asia and was tentatively identified as a new species of asia ethanollipciens (sp. Nov.). Strains S877, S1009, S1019 and S1023 were all identified as new species belonging to the new genus and were tentatively named as Zucharibacter floricola gen.nov., Sp.nov.

<2> production method of xylitol and D-xylulose

Xylitol and / or D-xylulose are capable of producing xylitol or D-xylose from glucose in a suitable medium so that xylitol or D-xylose, or both, accumulate in the medium. Can be produced by culturing microorganisms with and collecting xylitol and / or D-xylose from the medium.

The microorganism is not particularly limited as long as it has the ability to produce xylitol or D-xylose from glucose, and specific examples thereof include the aforementioned strains P528, S877, S1009, S1019 and S1023. Microorganisms belonging to the same species or the same genus as the above-mentioned strains and having the ability to produce xylitol or D-xylose from glucose can be used in the present invention. Examples of such microorganisms are, for example, a nucleotide sequence homologous to a nucleotide sequence in terms of molecular taxonomy based on the nucleotide sequence of SEQ ID NO: 1 or 16S rRNA sequence, or a molecular taxonomy based on the nucleotide sequence of SEQ ID NO: 2 or a 16S rRNA sequence. Flanks the microorganism belonging to the Acetobacteraceae family having a 16S rRNA gene comprising a nucleotide sequence homologous to a nucleotide sequence and having the ability to produce xylitol or D-xylose from glucose. In particular, mention may be made of microorganisms belonging to the genus Asia or Zucarybacter, more particularly asia ethanol lipasiens or Zucarybacter floricola.

The target product produced by the method of the present invention may be either xylitol or D-xylulose, or both.

According to the present invention, UV exposure, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment from ethyl microbial strains with the ability to produce xylitol or D-xylose from glucose, ethyl Certain mutant strains obtained by methanesulfonate (EMS) treatment, nitrous acid treatment and acridine treatment, or genetic engineering techniques such as genetic recombination or genetically modified strains obtained by cell fusion, and the like can also be used.

The medium for culturing the above-mentioned microorganisms may be a conventional medium containing a common carbon source, nitrogen source, inorganic ions, and optionally organic nutrients. Although the microorganisms of the present invention grow under high osmotic conditions, they can also grow under normal osmotic conditions. For example, strain P528 grows under normal osmotic conditions.

As the carbon source, carbohydrates such as glucose, alcohols such as glycerol and organic acids can be suitably used. In a preferred aspect observed in known methods for the production of xylitol, for example, the production of xylitol from pentitols such as D-xylose or D-arabitol, fructose and sucrose and Preference is given to disaccharides such as hexose, sucrose and lactose, and polysaccharides such as starch. These materials are used in the medium in amounts of 10 to 60%, preferably 20 to 50%, as the main carbon source. These carbon sources may be added to the medium at one time or in portions over time.

As the nitrogen source, ammonia gas, aqueous ammonia and ammonium salts are used. As inorganic ions, magnesium ions, phosphate ions, potassium ions, iron ions and manganese ions are used when necessary. As organic nutrients, substances containing organic nutrients such as vitamins, amino acids, and liver extracts, yeast extracts, malt extracts, peptones, meat extracts, corn liquor and casein breakdown products are optionally used.

Culture conditions are also not particularly limited. However, the microorganism may be cultured at a limited pH and temperature selected in the pH range of 5-8 and in the temperature range of 25-40 ° C. Cultivation is carried out under aeration conditions by agitation or shaking for aeration. For the incubation period, the microorganisms are preferably incubated until the main carbon source is consumed and is generally 3 to 8 days.

As described above, xylitol and / or D-xylose produced in the medium during this culture is collected separately from the culture in a conventional manner. In particular, for example, the solid material can be removed from the culture by centrifugation or filtration, decolorizing the residual solution and desalted using activated carbon or ion exchange resins, xylitol and / or D-xylul Rose is crystallized from solution. The separation and collection process of xylitol and / or D-xylulose from the culture is much easier than the separation from plant material hydrolyzate because of the low content of impurities.

The produced D-xylulose can be converted to xylitol by hydrogenation, which can be carried out by known methods.

<3> Ethanol production method

Ethanol can be produced by culturing microbial strain P528 (FERM BP-6751) in a medium containing glucose so that ethanol accumulates in the medium and collecting ethanol from the medium. Microorganism having, besides strain P528, a 16S rRNA gene comprising a nucleotide sequence homologous to a nucleotide sequence in terms of molecular taxonomy based on the nucleotide sequence of SEQ ID NO: 1 or the 16S rRNA sequence, and having the ability to produce ethanol from glucose, or Mutant strains thereof can be similarly used for ethanol production.

Media and culture conditions may be similar to those described above for the method for producing xylitol and D-xylulose. Ethanol produced in the medium can be purified by concentrating in the same manner as used for general ethanol fermentation.

The invention will be explained in more detail with reference to the following examples. However, the present invention is not limited to these examples.

In the examples, the xylitol and D-xylose produced are analyzed by high performance liquid chromatography (HPLC) under the following conditions.

Column: Sodex SC1211 [product of Showa Denko]

Mobile phase: 50 ppm of 50% acetonitrile / 50% Ca-EDTA aqueous solution

Flow rate: 0.8 ml / min

Temperature: 60 ℃

Detection: RI Detector

Example 1

Isolation of Microorganisms Producing Xylitol or D-Xylose

First, osmotic microorganisms are collected from nature by fortified culture. Medium containing 20% D-glucose, 1% yeast extract (manufactured by Difco) and 0.1% urea is introduced into the test tube in an amount of 4 ml each and sterilized at 120 ° C. for 20 minutes. Soil samples collected from various regions are inoculated into the medium and incubated with shaking at 30 ° C. for 4-7 days. If bacterial growth is observed, the cultures are plated on agar plates of the same composition and incubated at 30 ° C. for 1 to 3 days. Next, the colonies formed are picked up.

Next, about 3000 strains of osmotic bacteria obtained as described above were incubated at 30 ° C. for 5 days in a medium containing 20% (w / v) D-glucose, 0.1% urea and 0.5% yeast extract. The medium is analyzed by HPLC to screen for strains with xylitol or D-xylulose production capacity. As a result, five bacterial strains isolated from the soil collected from the banks of the Tama river in Kawasaki City, Kanagawa, were found to have the ability to produce xylitol from glucose. These strains are named P528, S877, S1009, S1019 and S1023, respectively. These five strains were designated in this order under the personal numbers AJ14757, AJ14758, AJ14759, AJ14760 and AJ14761, and were deposited on 18 June 1998 by the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology. 305-8566, Ikurakiken, Tsukuba-shi, Higashi-chome, Japan) in this order and deposited with the accession numbers FERM P-16848, FERM P-16849, FERM P-16850, FERM P-16851 and FERM P-16852 in 1999. The original deposit was transferred to the International Deposit under the Budapest Treaty on 14 June and deposited with Accession Numbers FERM BP-6751, FERM BP-6752, FERM BP-6753, FERM BP-6754 and FERM BP-6755.

Example 2

Production of Xylitol and D-Xylose from Glucose

Medium containing 0.5% yeast extract (manufactured by Difco) and 0.1% urea (pH 6.0) is introduced into a 500 ml Sakaguchi flask in an amount of 50 ml and sterilized by heating at 120 ° C. for 20 minutes. Separately sterilized glucose is added to this medium so that the medium contains 20% (w / v) glucose. Strains P528, S877, S1009, S1019 and S1023 are each inoculated into this medium and incubated with shaking at 30 ° C. for 5 days. The bacterial cells are then removed by centrifugation and xylitol and D-xylose formed in the medium are analyzed by HPLC. The results are shown in Table 10.

Production of Xylitol and D-Xylose Strain Concentration of produced xylitol (g / l) Concentration of produced D-xylose (g / l) P528 5.3 3.3 S877 1.9 9.7 S1009 1.6 9.0 S1010 1.7 9.2 S1023 1.5 5.0

Example 3

Molecular taxonomic analysis of strains P528, S877, S1009, S1019 and S1023

Strains P528 and S877 are analyzed in terms of molecular taxonomy by nucleotide sequence analysis of 16S rRNA in a conventional manner.

The bacterial cell suspension of each strain is treated with protease at 60 ° C. for 20 minutes, then heated in boiling water for 5 minutes and centrifuged. The obtained supernatant is used directly as a template for PCR.

this. 30 cycles of PCR were performed in a conventional manner using conventional primers corresponding to positions 8-27 and 1492-1515 of the 16S rRNA of E. coli (SEQ ID NO: 6 and SEQ ID NO: 7), and the product Is collected by PEG precipitation. PCR products are sequenced by fluorescent cycle sequencing and reaction products are analyzed by DNA sequencer (Pharmacia). The nucleotide sequences measured are shown in SEQ ID NO: 1 (strain P528) and SEQ ID NO: 2 (strain S877). No sequences corresponding to these nucleotide sequences were found in the database. The bacterial group with the closest nucleotide sequence of the 16S rRNA gene for each strain is bacteria belonging to the genus Gluconobacter and genus Acetobacter.

The obtained nucleotide sequence data is processed by GENETYX (Software Development, Tokyo), and multiple alignment and evaluation distance calculations are performed with CLUSTAL W on the obtained sequences and homologous sequences obtained from the database (13 kinds) The 16S rRNA gene sequence of the acetic acid bacterium of is considered a currently valid name). The obtained PHYLIP format data is read and processed by the Tree View to prepare a molecular phylogenetic tree. This result is shown in FIG. The alignment of 16S rRNA of xylitol producing bacteria is shown in FIGS. 2 and 3. The thirteen kinds of acetic acid bacteria mentioned above are mentioned below.

Gluconobacter asaii

Gluconobacter cerinus

Gluconobacter frateurii

Gluconobacter oxydans subsp.oxydans

Acetobacter aceti

Acetobacter pasteuranus

Acetobacter methanolicus

Gluconobacter europaeus

Gluconobacter xylinus subsp.xylinus

Gluconobacter intermedicus

Gluconobacter hansenii

Gluconobacter liquefaciens

Gluconobacter diazotrophicus

Rhodophila globiformis

As a result, known strains showing a close evaluation distance in relation to strain P528 are Gluconobacter intermedicus, Gluconobacter liquifaciens, Acetobacter aceti, Acetobacter methanolicus and Acetobacter pasteurianus, Its evaluation distance is 0.0345, 0.0359, 0.0403, 0.0419 and 0.0499, respectively, and the homology of the 16S rRNA gene is 96.5%, 96.3%, 96.0%, 95.9% and 95.1%, respectively. In addition, known strains showing a close evaluation distance in relation to strain S877 are Gluconobacter cerinus and Gluconobacter oxydans, whose evaluation distances are 0.0622 and 0.0629, respectively, and the homology of the 16S rRNA genes is 94.0% and 93.9%, respectively. to be. Although strain P528 is included in the population of the genus Acetobacter, it is far from three strains of known species and is therefore considered to be a new species. Acetobacter methanolicus has also been reported to belong to another genus (Axidocmonas genus). If Acetobacter methanolicus is considered to belong to another genus, strain P528 may belong to the new genus because this strain is located outside the population of the genus Acetobacter.

On the other hand, strain S877 is located outside the population of the genus Gluconobacter and is far from any known species belonging to the genus Gluconobacter. The evaluation distance from strain S877 to the closest strain (Glunobacter cerinus) is 0.066, which is significantly greater than the distance between the genus Gluconobacter and genus Acetobacter (0.044). Therefore, this strain is fully considered to belong to the novel genus.

When partial nucleotide sequences of the 16S rRNAs of strains S1009, S1019 and S1023 were measured (SEQ ID NOS: 3 to 5, respectively), they were found to be the same species since they exhibit substantially the same sequence as that of strain S877.

From the molecular taxonomic analysis described above and the phenotype shown in Table 9, strain P528 has been identified as a new species belonging to the genus Asia, and has been tentatively named asia ethanolifaciens sp. Nov. Strains S877, S1009, S1019 and S1023 were all identified as microorganisms of a new species belonging to the genus New and have been tentatively named as Zucharibacter floricola gen.nov., Sp.nov.

Example 4

Production of Xylitol and D-Xylose from Glucose

A medium containing 0.2% ammonium acetate, 0.3% potassium dihydrogenphosphate, 0.05% magnesium sulfate heptahydrate, 0.5% yeast extract (manufactured by Difco) and 4% calcium carbonate was introduced into a 500 ml sakaguchi flask in an amount of 50 ml and 120 Sterilize by heating at 20 ° C. for 20 minutes. Separately sterilized glucose is added to the medium so that the medium contains 20% (w / v) glucose. Strain P528 is inoculated into this medium and incubated at 30 ° C. for 4 days with shaking. Next, after bacterial cells are removed by centrifugation, xylitol and D-xylose formed in the medium are measured by HPLC. As a result, it was found that 6.4 g / L xylitol and 17.5 g / L D-xylulose were formed.

Example 5

Biochemical and Physical Properties of Strains P528, S877, S1009, S1019, and S1023

(1) Analysis of GC content of quinones and DNA

The GC content of the quinone and DNA of the above-mentioned strains was determined by conventional methods (Saikingaku Gijutsu Sosho (Library of Techniques in Bacteriology), vol. 8 "Method for Microbial Identification Following New Taxonomy", pp. 61-73, pp. 88-97, Saikon Shuppan, Japan] and analyzed by high performance liquid chromatography (HPLC). The results are shown in Table 11.

Quinone type and GC content of DNA Strain P528 S877 S1009 S1019 S1023 Quinone UQ-10 UQ-10 UQ-10 UQ-10 UQ-10 GC (%) 56.5 52.3 52.3 51.9 52.9 UQ: Ubiquinone

(2) Acid production from various carbon sources

Each of the aforementioned strains is cultured in a medium containing various carbon sources (1%) and the presence of acid formed is measured. Strains are precultured at 28 ° C. in YPG medium for 1 day, bacterial cells are washed with 0.5% yeast extract solution, inoculated in YPC medium and incubated at 28 ° C. for 4-7 days. The production of acid is then measured by the color change (red to yellow) of the pH indicator in the medium.

YPG medium is prepared as follows. The medium containing 1% yeast extract (manufactured by Difco) and 1% peptone is sterilized by heating at 120 ° C. for 20 minutes. To this medium, separately sterilized D-glucose is added in an amount containing 7% D-glucose of the medium.

YPC medium is prepared as follows. The medium containing 0.5% yeast extract (manufactured by Difco), 0.012% bromocresol purple and 1% of various carbon sources is sterilized by heating at 120 ° C. for 20 minutes.

The results are shown in Table 12.

Acid formation from various carbon sources Strain P528 S877 S1009 S1019 S1023 Xylose + - - - - Arabinose + - - - - Glucose + + + + + Galactose + - - - - Mannose + - + + + Fructose + + - - - Sorboz ± - - - - Sucrose ± + + + + Maltose - - - - - Rhamnose + - - - - Glycerol ± - - - - Mannitol ± + + + + Sorbitol ± - - - - Lactose + - - - - Starch - - - - - ethanol - - - - - +: Acid production, ±: weak acid production,-: no acid production

(3) Effect of NaCl Addition on Growth

The effect of NaCl addition on the growth of the strains described above is tested by culturing in YPM medium. Acetobacter aceti strain NCIB 8621 as the above-mentioned strain and the control strain was pre-incubated for one day at 28 ° C. in the above-described YPG medium, and the bacterial cells were washed with YPG medium without D-glucose and D-glucose Suspension in YPG medium not added. The obtained bacterial suspension is inoculated (1.6% v / v) in YPM medium in which NaCl is added at one of various concentrations and incubated with shaking at 28 ° C. for 2 days. Then, the turbidity of the medium is measured by the photometer ANA-75A obtained from Tokyo Koden (OD 660 nm) to measure growth.

YPM medium is prepared as follows. The medium containing 1% yeast extract (manufactured by Difco), 1% peptone and 1% mannitol is sterilized by heating to 120 ° C. for 20 minutes.

This result is shown in FIG. Strain P528 grows well in medium supplemented with up to 2% NaCl, ie exhibits NaCl resistance.

(4) consumption of acetic acid and lactic acid

The above-mentioned strains are cultured in YG medium supplemented with acetic acid or lactic acid to test the consumption of acetic acid and lactic acid.

Acetobacter aceti strain NCIB 8621 as the above-mentioned strain and a control group is pre-cultured in YPG medium as described above with shaking at 28 ° C. for 1 day. The obtained preliminary medium is inoculated (1.6% v / v) in YG medium added with 1% acetic acid or lactic acid and incubated at 28 ° C. for 7 days. The consumption of acetic acid and lactic acid is tested by sampling of the medium over time. Measurement of acetic acid and lactic acid is carried out by HPLC under the following conditions.

Column: ULTTRON PS-80 from Shinwa Kagaku Kogyo

Mobile phase: Perchloric acid solution (pH 2.1)

Flow rate: 0.9ml / min

Temperature: 60 ℃

Detection: UV Detector (210 nm)

A YG medium to which acetic acid or lactic acid was added was prepared as follows. The medium containing 1% yeast extract (manufactured by Difco) and 1% acetic acid or lactic acid is sterilized by adjusting to pH 6.0 and heating to 120 ° C. for 20 minutes. Separately sterilized D-glucose is added to the medium in an amount such that the medium contains 7% D-glucose.

The results are shown in Table 13 (data indicates the amount consumed (%)). Strains P528, S877, S1009, S1019 and S1023 all exhibit lactic acid consuming ability, while they have weak or substantially no acetic acid consuming ability.

Consumption of acetic acid and lactic acid Strain P528 S877 S1009 S1019 S1023 a. Aceti Acetic acid (%) 87.8 100.0 88.6 100.0 96.6 7.7 Lactic acid (%) 0.0 4.7 29.7 30.7 24.3 0.0 a. Aceti: Acetobacter Aceti strain NCIB8621

(5) Effect of Addition of Acetic Acid or Ethanol on Growth

The effect of acetic acid addition on the growth of the strains described above is tested in acetic acid added YG medium. The effect of ethanol addition on the growth of the strains described above is also tested in YPG medium with ethanol group addition.

The above-mentioned strains were preincubated at 28 ° C. for 1 day in the YPG medium, and each medium was inoculated (1.6% v / v) in YG medium to which lactic acid was added at various concentrations, and 10 days at 28 ° C. Incubate while shaking. The turbidity of the medium is then measured by photometer ANA-75A from Tokyo Koden (OD 660 nm) to measure growth.

All strains P528, S877, S1009, S1019 and S1023 grow well in medium to which 1% or less acetic acid or 3% or less ethanol was added. All strains do not grow in medium to which at least 4% acetic acid or at least 5% ethanol is added.

(6) acetic acid production and ethanol consumption

The above-mentioned strains are cultured in YPG medium added with ethanol to test acetic acid production and ethanol consumption. Acetobacter aceti strain NCIB8621 as the above-mentioned strain and a control group is preincubated at 28 ° C. for 2 days in the above-described YPG medium. Each medium is inoculated (1%, v / v) in YPG medium with 1% ethanol and incubated at 28 ° C. The concentrations of acetic acid and ethanol in the medium are tested by sampling over time of the medium. Measurement of acetic acid and ethanol concentrations is carried out with F-kit (Roche Dianostics).

The results are shown in FIGS. 5 and 6. Strains P528, S877, S1009, S1019 and S1023 all exhibit weak acetic acid production compared to the control bacterium Acetobacter aceti strain NCIB 8621 (FIG. Only shows data for strains P528 and S877). In addition, strains S877, S1009, S1019 and S1023 show no ethanol consumption in contrast to the control bacterium Acetobacter aceti strain NCIB 8621. Strain P528, in contrast, shows an increase in the amount of ethanol.

Example 6

Production of Ethanol by Strain P528

Strain P528 is incubated using YPG medium in a similar manner as described above and the ethanol concentration in the medium is measured over time. This result is shown in FIG. This strain P528 represents ethanol production.

According to the present invention, microorganisms having the ability to produce xylitol or D-xylulose by fermentation and methods for producing xylitol or D-xylulose using such microorganisms are provided.

Claims (15)

Has a 16S rRNA gene comprising a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 1 in terms of molecular taxonomy based on the nucleotide sequence of SEQ ID NO: 1 or the 16S rRNA sequence and produces xylitol or D-xylose from glucose A microorganism belonging to the Actetobacteracea family having the ability to do so. The microorganism of claim 1, wherein the microorganism belongs to the genus Asia. The microorganism of claim 2, which is an Asia ethanolifaciens strain. Has a 16S rRNA gene comprising a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 2 in terms of molecular taxonomy based on the nucleotide sequence of SEQ ID NO: 2 or the 16S rRNA sequence and produces xylitol or D-xylose from glucose Microorganisms with the ability to do so. The microorganism of claim 4, wherein the 16S rRNA gene comprises any one of the nucleotide sequences of SEQ ID NO: 3-5. The microorganism according to claim 4, which belongs to the genus Zucharibacter. The microorganism according to claim 4, which is a Zucharibacter floricola strain. Microbial strain P528 (FERM BP-6751) with the ability to produce xylitol or D-xylose from glucose. Microbial strain S877 (FERM BP-6752) with the ability to produce xylitol or D-xylose from glucose. Microbial strain S1009 (FERM BP-6753) with the ability to produce xylitol or D-xylose from glucose. Microbial strain S1019 (FERM BP-6754) with the ability to produce xylitol or D-xylose from glucose. Microbial strain S1023 (FERM BP-6755) with the ability to produce xylitol or D-xylose from glucose. Culturing a microorganism having the ability to produce xylitol or D-xylose from glucose in a medium suitable for accumulating xylitol or D-xylose in the medium, and xylitol from the medium. Or collecting D-xylulose. The method of claim 13, wherein the microorganism is the microorganism according to claim 1. Culturing the microbial strain P528 (FERM BP-6751) in a medium suitable for accumulating ethanol in the medium, and collecting ethanol from the medium.