JP2010035553A - PRODUCTION METHOD OF BIOMASS KEEPING alpha-GLUCAN CONVERTED FROM PLANT CELL WALL COMPONENT - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a production of biomass of bacteria massively stored as α-glucan by assimilating poorly used monosaccharide obtained by hydrolysis of plant cell wall components. <P>SOLUTION: Provided is a production method of biomass keeping α-glucan converted from plant cell wall components, wherein plant cell wall components are hydrolyzed to monosaccharides by treating with an acid or an enzyme hydrolyzing the plant cell wall components; in a culture solution containing the monosaccharides, specific bacteria converting the monosaccharides into α-glucan and keeping are cultivated to assimilate the monosaccharides; and α-glucan of ≥10% based on biomass dry weight of the bacteria is accumulated in the biomass of the bacteria. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a method for producing a microbial cell that retains α-glucan converted from plant cell wall components, and more specifically, a large amount of 'α-glucan of 10% or more per microbial dry weight of the bacterium, The present invention relates to a method for producing a microbial cell that retains α-glucan converted from a plant cell wall component, characterized in that the microbial cell is accumulated in the microbial cell.
Moreover, this invention relates to the method of manufacturing glucose from the microbial cell holding the alpha-glucan obtained from the said manufacturing method, Furthermore, the method of manufacturing ethanol from the said glucose.

When developing biomass utilization technology that does not compete with food, waste resources such as primary processing residues of agricultural products and food processing waste, and unused rice straw, rice husk, wheat straw, bagasse, weeds, lumber residues, thinned wood, etc. Development of effective utilization technology for resources and energy crops is the key. In these uses, it is important to develop technologies for utilizing cellulose, hemicellulose, and pectin that constitute plant cell walls.
Among the constituent sugars of the plant cell wall, hexose sugars such as glucose and fructose are likely to be substrates for fermentable bacteria such as yeast, and are used as raw materials for production of bioethanol and other various fermentation products. However, the plant cell wall contains various sugars such as arabinose, galacturonic acid, rhamnose, and xylose in addition to the above hexoses, which are mainly macromolecules such as 'hemicellulose' and 'pectin'. It exists as a component of

However, the utilization technique of these components is not fully developed.
For example, in the process of converting the constituent sugars of “hemicellulose” and “pectin” into ethanol, microorganisms having fermentability are known for “xylose” and “arabinose” which are constituent sugars of hemicellulose. However, the xylose-fermenting yeast Pichia stipitis has problems such as oxygen demand and low ethanol tolerance, and it has not been completed as a fermentation technology at commercial level. It's far away.
In addition, a method using a microorganism into which a gene responsible for the metabolism is introduced has been studied. For example, by introducing an ethanol metabolism gene into Escherichia coli that can assimilate various carbohydrates such as xylose and arabinose, an ethanol-fermenting Escherichia coli called KO11 has been produced and used in various businesses. In addition, many attempts have been made to improve the ethanol production efficiency by introducing genes responsible for the metabolic pathways of xylose and arabinose to high-concentration ethanol-resistant bacteria such as yeast and bacteria belonging to the genus Zymomonas.
However, when these genetic recombination technologies are applied, there is a concern that the equipment and management costs for a system for suppressing the release of recombinant microorganisms to the environment will be high.

At present, the use as a functional food material exists as the main usage of 'hemicellulose' and 'pectin'. For example, xylitol, xylo-oligosaccharides, arabinose, pectin oligosaccharides and the like have been developed and some have been put into practical use. However, the market is small, and it is not an outlet that can cover the amount of by-products from a large amount of biomass feedstock. Thus, development of an effective utilization technique of cell wall components mainly composed of hemicellulose and pectin is required.
In addition, there are uses that have low availability such as arabinose, galacturonic acid, rhamnose, and xylose, but are used for composting and methane fermentation. In particular, it is desirable to convert to substances that are consumed in large quantities throughout the year, such as food, feed, microbial nutrient sources, or chemical industry raw materials and fuels.
Under such circumstances, “α-glucan” such as glycogen is a polysaccharide carbohydrate composed of glucose, and is a substance that is enzymatically degraded by a large number of organisms including higher animals such as food, feed and It is a carbohydrate that can be expected to be used as a microbial nutrient source.
Further, α-glucan is decomposed not only by an enzyme but also by acid treatment, and can be converted to glucose. This indicates that glucan is highly useful in the production of various chemical industrial raw materials and fuels such as bioethanol, in which a fermentation system from glucose has been completed.
However, for this purpose, no technology has been developed to produce α-glucan by converting low-usage carbohydrates (hemicellulose or pectin), which are plant cell wall components, and development of effective utilization technology has not been carried out. Is expected.

Many microorganisms themselves producing α-glucan such as glycogen are known. For example, in addition to eukaryotes such as yeast, it is known that many bacteria such as Bacillus subtilis, Streptomyces coelicolor, Micropruina glycogenica, Mycobacterium smegmatis, Corynebacterium glutamicum, Escherichia coli, Mesorhizobium loti are produced.
Regarding these α-glucans derived from microorganisms, when cultured in a medium containing “glucose”, data has been obtained that accumulates up to about 8.4% or 9% in terms of the dry weight ratio of the cells ( For example, refer nonpatent literature 1 and 2.).
However, many of these microorganisms have a background in which basic research has been conducted as a key group of microorganisms for removing 'phosphoric acid' by the activated sludge method, and 'low-use monosaccharides derived from plant cell wall components. There has been no research associated with technology to convert '. Further, the amount of accumulation is insufficient in practical use.
There are also Escherichia coli and Micropruina glycogenica that have the ability to metabolize monosaccharides such as xylose and arabinose and accumulate glycogen (see, for example, Non-Patent Documents 1 and 3). A technique for accumulating a large amount of α-glucan that is recognized as being not studied. In addition, the accumulation characteristics of α-glucan when these microorganisms metabolize constituent sugars such as hemicellulose and pectin other than xylose and arabinose have not been reported.

Shintani, T., et al., Int. J. Syst. Evol. Microbiol., 50, 201-207 (2000) Seibold, G., et al., Microbiology, 153, 1275-1285 (2007) Govons. S., et al., J. Bacteriol., 97,970-972 (1969)

  The present invention solves the above-mentioned conventional problems, assimilates a low-usage monosaccharide obtained by hydrolyzing plant cell wall components, and accumulates a large amount of α-glucan as a bacterial cell It is a problem to manufacture.

  The inventor hydrolyzes a plant cell wall component into a monosaccharide by treating with an acid or an enzyme that hydrolyzes the plant cell wall component; and in a culture solution containing the monosaccharide, -Specific bacteria that convert and retain glucan, especially Enterobacter cancerogenus S24 (FERM P-21598) strain, Arthrobacter sp. 4P-01-A1 (FERM P-21807) strain, or Sporosarcina sp. N52 (FERM P-21808) strain, or a mutant thereof, and by assimilating the monosaccharide; It was found that “a large amount” of α-glucan of 10% or more per body dry weight can be accumulated in the cells of the bacteria, and the present invention has been completed.

The present invention relates to the following.
That is, the present invention according to claim 1 hydrolyzes a plant cell wall component into a monosaccharide by treating with an acid or an enzyme that hydrolyzes the plant cell wall component; and in a culture solution containing the monosaccharide. Culturing a bacterium belonging to the Proteobacteria class, Actinobacteria class, or Bacilli class, which has the property of assimilating the monosaccharide and converting it into α-glucan, and assimilating the monosaccharide; A method for producing a microbial cell that retains α-glucan converted from plant cell wall components, characterized in that 10% or more of α-glucan per dry weight of the bacterial cell is accumulated in the microbial cell of the bacterium. It is.
In the present invention according to claim 2, the monosaccharide is selected from the group consisting of D-xylose, L-arabinose, D-galacturonic acid, L-rhamnose, D-glucuronic acid, D-galactose, and D-mannose. The method for producing a microbial cell retaining α-glucan according to claim 1, which is at least one of the above.
Moreover, the present invention according to claim 3 converts the monosaccharide in the culture solution into α-glucan in an amount of 10% or more of the monosaccharide amount in terms of glucose. The present invention relates to a method for producing a microbial cell retaining the α-glucan.
Further, in the present invention according to claim 4, the concentration of the monosaccharide in the culture solution is 0.05% (w / v) or more and less than 0.5% (w / v). It is related with the manufacturing method of the microbial cell holding the alpha-glucan in any one of claim | item 1-3.
In addition, the present invention according to claim 5 hydrolyzes a plant cell wall component by treating with an acid or an enzyme that hydrolyzes the plant cell wall component, and the Proteobacteria class in a culture solution containing the hydrolyzate. , Culturing bacteria belonging to the Actinobacteria class or the Bacilli class and assimilating the hydrolyzate; by the above-mentioned bacteria, 10% or more of α-glucan per dry weight of the bacteria is contained in the bacteria It is related with the manufacturing method of the microbial cell holding the alpha glucan converted from the plant cell wall component characterized by making it accumulate | store.
Further, in the present invention according to claim 6, when the bacterium is selected from soil, by assimilating the monosaccharide, 10% or more of α-glucan per dry cell weight of the bacterium is obtained. The present invention relates to a method for producing a microbial cell retaining α-glucan according to any one of claims 1 to 5, which is selected using the property of accumulation in the microbial cell as an index.
Moreover, this invention of Claim 7 is a microbial cell holding the alpha-glucan in any one of Claims 1-6 whose said bacteria are bacteria which belong to Enterobacter genus, Arthrobacter genus, or Sporosarcina genus. Manufacturing method.
The present invention according to claim 8 is characterized in that the bacterium is Enterobacter cancerogenus S24 (FERM P-21598) strain, Arthrobacter sp. 4P-01-A1 (FERM P-21807) strain, or Sporosarcina sp. N52 (FERM P-21808) strain, or a mutant thereof, and relates to a method for producing a microbial cell retaining α-glucan according to any one of claims 1 to 7.
Moreover, this invention of Claim 9 is a microbe holding the alpha-glucan in any one of Claims 1-8 whose said plant cell wall component is what has hemicellulose and / or pectin as a main component. The present invention relates to a method for manufacturing a body.
Moreover, this invention of Claim 10 is a manufacturing method of the microbial cell holding the alpha-glucan in any one of Claims 1-9 whose said plant cell wall component is what has a xylan as a main component. , About.
Moreover, this invention of Claim 11 is a manufacturing method of the microbial cell holding the alpha-glucan in any one of Claims 1-10 whose said alpha-glucan has glycogen as a main component. , About.
In addition, the present invention according to claim 12 retains the α-glucan according to any one of claims 1 to 11, wherein the bacteria are cultured and then separated into solid and liquid to recover the cells as solids. The present invention relates to a method for producing bacterial cells.
Moreover, this invention of Claim 13 is related with the microbial cell holding the alpha-glucan obtained by the manufacturing method in any one of Claims 1-12.
In the present invention described in claim 14, after destroying the microbial cells holding the α-glucan according to claim 13, the α-glucan is converted into α-amylase, β-amylase, pullulanase, amyloglucosidase, α -The manufacturing method of glucose which hydrolyzes to glucose by processing with at least 1 or more enzyme among glucosidases.
Further, the present invention according to claim 15 is a method of treating α-glucan by treating the bacterial cell retaining α-glucan according to claim 13 at a temperature of 60 ° C. or higher with dilute hydrochloric acid or dilute sulfuric acid. The present invention relates to a method for producing glucose, which is hydrolyzed to glucose.
Moreover, this invention of Claim 16 is related with the manufacturing method of ethanol characterized by using the glucose obtained by the manufacturing method in any one of Claim 14 or 15 as a raw material of ethanol fermentation. Is.
In addition, the present invention described in claim 17 is obtained by the α-glucan contained in the microbial cells holding the α-glucan according to claim 13 or the production method according to any one of claims 14 or 15. Food, feed or microbial nutrient source containing the glucose produced.

The present invention comprises assimilating a 'low-utility' monosaccharide obtained by hydrolyzing plant cell wall components to produce bacterial cells that have accumulated 'a large amount' as α-glucan. Make it possible.
Next, the present invention relates to a bacterium obtained by assimilating a hydrolyzate composed of a 'low-utility' monosaccharide obtained by hydrolyzing plant cell wall components and accumulating 'a large amount' as α-glucan. It is possible to produce microbial cells.
In addition, the present invention makes it possible to produce glucose from plant cell wall components, and further to produce ethanol from plant cell wall components.
In addition, according to the present invention, highly useful glucan-carrying cells can be recovered by solid-liquid separation while decomposing the organic matter in the waste liquid after ethanol fermentation to reduce the BOD of the waste liquid.
Moreover, it becomes possible to obtain a glucan holding | maintenance microbial cell, reducing BOD as a part of waste liquid processing process, especially activated sludge processing process.

In Example 2, it is a figure which shows the measurement result of the amount of glucose liberated by the enzyme process. In Example 11, it is a figure which shows the measurement result of the amount of glucose liberated by the enzyme process. In Example 15, it is a figure which shows the measurement result of the amount of glucose liberated by the enzyme process. In Example 18, it is a figure which shows the measurement result of a dry microbial cell weight and a microbial cell glucan retention amount. In Example 18, it is a figure which shows the measurement result of the quantity of each saccharide | sugar in a culture solution.

  The present invention hydrolyzes a plant cell wall component into a monosaccharide by treating with an acid or an enzyme that hydrolyzes the plant cell wall component; and assimilates the monosaccharide in a culture solution containing the monosaccharide. By culturing a specific bacterium having the property of being converted to α-glucan and retaining it, and assimilating the monosaccharide; 'large amount' of α-glucan of 10% or more per dry weight of the bacterium Is accumulated in the microbial cells of the bacterium, and relates to a method for producing microbial cells retaining α-glucan converted from plant cell wall components.

“Bacteria that retain α-glucan” refers to cells that have accumulated α-glucan in the bacterial body or in the extracellular matrix. In the fraction 'recovered as a solid content at the time of solid-liquid separation', it refers to the bacterial cells accumulating α-glucan.
'α-glucan' is a polysaccharide sugar composed of 'glucose' as a constituent sugar, and accumulates not only humans and many animals and plants, but also bacteria and fungi, and has a corresponding hydrolase. . For this reason, it is expected to be a carbohydrate that is highly useful for food, feed, microbial nutrients, and the like.

As the main α-glucan, starch, glycogen, pullulan, dextran and the like are known.
Of these, glycogen is expected to be highly available because it is amorphous and extremely biodegradable. Natural starch has high crystallinity, and can be used after heat denaturation and gelatinization to lower crystallinity and improve enzyme degradability.
Therefore, the α-glucan accumulated in the microbial cells in the present invention is particularly preferably glycogen from the viewpoint of high availability.
Cellulose, β- (1 → 3) -glucan, β- (1 → 3)-(1 → 6) -glucan, etc. also have “glucose” as a constituent sugar. ”And is low in utility in terms of the corresponding hydrolase and the like. Therefore, the present invention does not target bacterial cells that retain these β-glucans.

As the “bacteria” used as a microbial cell that retains α-glucan in the present invention, Proteobacteria class (class Proteobacteria), Actinobacteria class (class Actinobacteria class) having the property of assimilating monosaccharides described later and converting them into α-glucan and retaining them. ), Or a bacterium belonging to the class Bacilli.
As the bacteria that can be used in the present invention, bacteria belonging to the genus Enterobacter, Arthrobacter, or Sporosarcina are preferably used.
Furthermore, Enterobacter cancerogenus S24 (FERM P-21598) strain, Arthrobacter sp. 4P-01-A1 (FERM P-21807) strain, or Sporosarcina sp. N52 (FERM P-21808) strain, or a mutant thereof, It is desirable to be.
In addition, the microorganisms themselves that produce α-glucan are extremely diverse, and many bacteria are known in addition to a group of glycogen-accumulating microorganisms including unidentified microorganisms that are problematic in the recovery process of phosphoric acid by the activated sludge method. However, nothing has been reported so far that satisfies both “assimilability of monosaccharide obtained by hydrolyzing plant cell wall components” and “mass accumulation capacity of glycogen” at the same time.

The “plant cell wall component” refers to a polysaccharide mainly constituting the plant cell wall, and examples thereof include cellulose, hemicellulose, and pectin. In the present invention, particularly, hemicellulose and pectin containing a large amount of low-usage carbohydrates (monosaccharides) are targeted. ("Hemicellulose" is a generic term for polysaccharides constituting the plant cell wall excluding cellulose and pectin.)
Examples of 'hemicellulose' containing a large amount of low-usage carbohydrates (monosaccharides) include xylan (including glucuronoxylan, glucuronoarabinoxylan, arabinoxylan), glucomannan, xyloglucan, and the like. In the present invention, specifically, xylan can be used.
Examples of 'pectin' containing a large amount of low-usage carbohydrates (monosaccharides) include homogalacturonan, rhamnogalacturonan, arabinogalactan, arabinan, galactan and the like.

These plant cell wall components (hemicellulose, pectin) are low-usage carbohydrates such as D-xylose, L-arabinose, D-galacturonic acid, L-rhamnose, D-glucuronic acid, D-galactose, D-mannose ( Monosaccharide) as a constituent sugar in a large amount.
In particular, 'L-arabinose' is contained in many types of hemicellulose and pectin and has a large content. In addition, it is advantageous in that it exists widely in monocotyledonous cell walls, dicotyledonous cell walls and woody cell walls.

In the present invention, as a raw material for plant cell wall components, cell wall components of all eukaryotic green plants such as angiosperms, gymnosperms, ferns, moss plants, and algae (Aosa algae, green algae, axle algae) can be used. In the vascular plant, all organs such as roots, stems and leaves can be used. In addition, any crops such as resource crops and weeds can be used regardless of their usefulness as resources, but it is preferable to use low-utility plant species, organs, processing residues of crops, and the like.
For example, thinned wood and forest residue from broad-leaved trees, conifers, non-edible parts of grains (stems, rice husks, cores, roots, etc.), bamboo, and processing residues derived from crop crops (for example, , Rice straw, wheat straw, sugarcane bagasse, corn stover, starch pomace, sugar beet pomace, ethanol fermentation residue and distillate waste, vegetable residue, floating / suspension fraction in juice production process, vegetable-derived cooking waste , Etc.), non-food standard parts, weeds, dicotyledonous stems and leaves, non-vascular plants, algae and seaweeds grown in fresh water, and microalgae such as chlorella.

The plant cell wall component can be assimilated by the bacterium after being hydrolyzed to a “monosaccharide” by treating with “acid” or “an enzyme that hydrolyzes the plant cell wall component” (plant cell wall component) Hydrolysis step).
In this hydrolysis step, even when the plant cell wall component cannot be completely hydrolyzed to a monosaccharide, the hydrolyzate is in a state of 'a sugar of a size that can be decomposed into a monosaccharide by the enzyme of the bacterium itself'. If so, the bacteria can be assimilated. Preferably, it is desirable to hydrolyze to a “monosaccharide” state by the hydrolysis treatment in this step.
Moreover, after performing hydrolysis by mild “acid treatment”, galacturonic acid having low stability against thermal acid can be efficiently recovered by further performing “enzyme treatment” hydrolysis.

Here, the acid treatment when the plant cell wall component is hydrolyzed can be performed using an organic acid aqueous solution such as formic acid, acetic acid aqueous solution or citric acid, dilute hydrochloric acid, dilute sulfuric acid or dilute nitric acid. Specifically, for example, it can be carried out using dilute sulfuric acid of about 0.05% to 60%, desirably about 0.5% to 10%.
In addition, when performing the said acid treatment, the said process can be made efficient by heating at 60 degreeC or more. Furthermore, by performing the enzyme treatment after the acid treatment, the acid treatment conditions can be moderated, and the amount of acid used and pH change can be suppressed.

The treatment with an enzyme that hydrolyzes the plant cell wall component is performed by xylanase, β-glucanase, β-D-xylosidase, α-L-arabinofuranosidase, α-D-galactosidase, β-D-galactosidase, α -Using enzymes such as L-glucuronidase, β-mannosidase, β-mannanase, glucomannanase, polygalacturonase, pectinase, endoarabinanase, feruloyl esterase, acetyl xylan esterase, α-L-rhamnosidase, pectin methyl esterase Can be done.
Which of these enzymes is used depends on the sugar composition and structural characteristics of the raw material. For example, in xylan, xylanase, β-D-xylosidase, α-L-arabinofuranosidase, α-L- By using glucuronidase or the like, it can be carried out efficiently.
When hydrolyzing by the enzyme treatment, a method of “using an enzyme produced by the bacterium itself” or a method of “adding an enzyme produced by another organism” can be considered.
In the case of using the method “using an enzyme produced by the bacterium itself”, an exogenous degrading enzyme gene can be introduced into the bacterium to produce a degrading enzyme.
Moreover, when performing by the method of 'adding an enzyme produced by another organism', the efficiency of hydrolysis can be improved by subjecting the plant cell wall component to pretreatment (for example, weak alkali treatment) in advance. In addition, by treating with weak alkali as a pretreatment, it is possible to remove acetyl groups, methyl groups, and the like that modify cell wall components, and to cleave bonds between molecules such as lignin and ferulic acid and polysaccharides.

  In addition, when performing the said process (plant cell wall component hydrolysis process) by an enzyme process, it is also possible to carry out as a "parallel multiple reaction" by the same reaction system as the following "culture utilization process".

  The 'monosaccharide' hydrolyzed from the plant cell wall component obtained after the above step is assimilated by culturing the bacterium in a culture solution containing the monosaccharide (culture assimilation step), A large amount of 'α-glucan can be accumulated in the cells of the bacteria.

In this step, the bacterium can be assimilated by culturing the bacterium in a culture solution 'containing the monosaccharide'.
As the culture solution used in this step, any culture solution can be used as long as it is suitable for the growth of the bacteria. For example, M9 medium, YE medium, LB medium, YP medium and the like can be used.
In addition, the culture conditions in this step can be any conditions as long as they are suitable for the growth of the bacteria. For example, it can carry out efficiently on the conditions of 10-45 degreeC, 120-200 rotation speed / min, 8-72 hours.

In addition, as a density | concentration of the "monosaccharide" which is a substrate contained in the culture medium used at this process, it can carry out at a high density | concentration as long as a microbial cell can grow.
The substrate concentration is preferably “high concentration” as long as growth is possible because the total retained amount of α-glucan that can be recovered increases. However, the higher the substrate concentration, the lower the growth efficiency and the lower the conversion efficiency to α-glucan per substrate.
Therefore, from the viewpoint of 'effective use of the substrate', it is desirable that the substrate concentration is less than about 1% (w / v), preferably less than 0.5% (w / v), more preferably 0. .2% (w / v) or less is desirable. In view of the total amount of collected recovered at the same time, it is preferably 0.05% (w / v) or more.

The bacterium after the main culture assimilation step has an α-glucan accumulated and retained in the microbial cells at a 'retention rate' of 10%, preferably 20% or more per dry weight of the bacterium. Is.
In addition, the bacterium after the main culture assimilation step is the “conversion rate” of the “monosaccharide” contained in the culture solution into α-glucan (the amount of glucose released from the accumulated α-glucan / before the culture). The amount of arabinose in the culture solution × 100) is preferably 10% or more.
Here, “retention rate” (retention amount) and “conversion rate” of α-glucan are determined by measuring the amount of glucose, which is a carbohydrate (monosaccharide) constituting α-glucan, and converting it from the value. (Calculated in terms of glucose).

After completion of the culture assimilation step, the obtained microbial cells are separated into solid and liquid, and the microbial cells can be recovered as a solid content by removing the culture solution. (Cell recovery process)
As a method of solid-liquid separation, any method can be used. For example, it can be performed by centrifugation.

By passing through the above steps, a “bacterial body that retains“ a large amount ”of α-glucan converted from plant cell wall components” can be produced.
By producing such α-glucan-carrying cells, it is possible to convert a low-usage monosaccharide in a solution into highly-usable α-glucan and concentrate it.
Normally, when concentrating a low molecular weight carbohydrate such as a monosaccharide, it is performed by heating evaporation or vacuum concentration, or by filtration concentration using an RO membrane or the like, but the method of the present invention is publicly known. Since it can be collected by methods used in microbiological research and bioprocesses, such as natural sedimentation, centrifugation, membrane separation, etc., bacterial cells in which substrate conversion products have accumulated can be easily concentrated and collected.

The α-glucan-carrying bacterial cells can be used in the form of suspensions, powders, bacterial cell pellets, etc., or in the form of crushed materials or hydrolysates, and can be used as food, feed or microbial nutrient sources as carbohydrate sources. it can.
Since α-glucan retained in the cells can be decomposed by digestive enzymes possessed by humans, animals, and microorganisms, it is extremely useful.
In addition, α-glucan retained in the cells can be hydrolyzed by destroying the cell walls physically, self-dissolving after cell death, or enzymatically or chemically destroying them. It can be made efficient.
When used in food, it is important that the bacteria used in the present invention do not produce harmful substances. Moreover, as above-mentioned, the utility as nutrient sources other than sugar can be considered.

Bacteria that retain α-glucan obtained in the above steps in a “large amount” can efficiently generate “glucose” by hydrolyzing the retained α-glucose (glucose generating step). .
This glucose production step is carried out by i) a method of treating with the enzyme that hydrolyzes the α-glucan, or ii) a method of heat-treating the cells holding the α-glucan using dilute hydrochloric acid or dilute sulfuric acid. be able to.
Alternatively, i) and ii) can be combined and performed.

i) 'The method of treating with the enzyme that hydrolyzes α-glucan' is carried out by destroying the cells holding the α-glucan and then treating the enzyme that hydrolyzes the α-glucan. it can.
In the present method, the destruction of the microbial cells retaining the α-glucan is, for example, autolysis treatment, lysis enzyme treatment such as lysozyme, surfactant treatment, acid or alkali lysis treatment, osmotic pressure treatment, 60 ° C. The above-described methods such as high-temperature treatment, high-pressure treatment, freeze crushing treatment, ultrasonic crushing treatment, and physical crushing treatment using a bead mill can be used. The disrupted bacterial cell destruction product can be recovered as an “α-glucan-containing fraction” by recovering the precipitate portion by centrifugation or the like. The collected fraction can be suspended and dissolved in a reaction solution to carry out an enzyme reaction.
In the present method, α-amylase, β-amylase, pullulanase, amyloglucosidase, α-glucosidase and the like can be used as the enzyme that hydrolyzes α-glucan. Preferably, it is preferable to use the combination of 'α-amylase and pullulanase' or 'α-amylase and amyloglucosidase' at the same time, and furthermore, the combination of three types of 'α-amylase, pullulanase and amyloglucosidase' at the same time. It is more preferable to use it.
The reaction conditions for the enzyme can be a reaction solution suitable for the reaction of each enzyme, and temperature and time conditions. Specifically, when three types of combinations of 'α-amylase, pullulanase and amyloglucosidase' are used at the same time, for example, using a sodium acetate buffer having a pH of around 4.5 to 6.5 at 37 to 50 ° C, The reaction can be carried out for minutes to 24 hours.

ii) “The method of heat-treating the bacterial cells holding the α-glucan with dilute hydrochloric acid or dilute sulfuric acid” is performed at 60 ° C. or higher, preferably 100 ° C. with the dilute hydrochloric acid or dilute sulfuric acid. It can be performed by treating at a temperature of ° C or higher.
The concentration of dilute hydrochloric acid or dilute sulfuric acid can be about 0.01% to 9%. Moreover, as processing time, it can carry out in about 10 minutes-120 minutes.

By passing through this glucose production | generation process, the solution (hydrolysis solution) which produced | generated glucose can be obtained from the alpha-glucan currently hold | maintained at the said microbial cell.
The glucose contained in the solution can be used as a food, feed or microbial nutrient source. Further, it can be used for various fermentation processes using glucose as a raw material, in particular, ethanol fermentation using yeast.

Ethanol fermentation by yeast can be performed by any method as long as it uses the obtained glucose.
Specifically, ethanol can be produced by adding the hydrolysis liquid (preferably an enzyme reaction liquid) obtained in the glucose production step and performing ethanol fermentation with yeast.
In addition, when the hydrolysis liquid obtained at the said glucose production | generation process is the said enzyme reaction liquid, after inactivating an enzyme (for example, after performing a heat processing and an acid treatment above 70 degreeC), it depends on yeast. It is desirable to perform ethanol fermentation.
In addition, when the hydrolyzate obtained in the glucose production step is treated with dilute sulfuric acid or dilute hydrochloric acid, it is desirable to carry out ethanol fermentation with yeast after neutralization of acid or separation and recovery of glucose. .

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these Examples.

[About Enterobacter cancerogenus S24 (FERM P-21598) strain]
<Example 1> Selection and identification of S24 strain (1) Selection of strains Utilizing 'monosaccharide' obtained by hydrolyzing 'plant cell wall component' with acid or enzyme to accumulate in large quantities as α-glucan In order to select microorganisms having the ability, the microorganisms having the ability to assimilate arabinose and accumulate in large quantities as α-glucan were selected.
In addition, arabinose is one of the monosaccharides obtained in large quantities when the “plant cell wall component” is hydrolyzed with an acid or an enzyme.
First, 0.1 mg of a soil sample is suspended in 1 ml of sterilized water, diluted as appropriate, and placed on an M9 agar medium containing 0.2% arabinose (the composition of the M9 medium is shown in Table 1) and cultured at 30 ° C. overnight. did. Subsequently, the colonies obtained on the agar medium after the cultivation were picked with a toothpick and inoculated into an M9 liquid medium containing 1% arabinose, and cultured at 30 ° C. and 120 rpm for 14 hours.
Thereafter, 0.1 ml of the obtained “bacterial cell culture solution” was taken and centrifuged at 7000 × g for 5 minutes to remove the medium, and then the bacterial cell as a precipitation part was obtained.
0.1 ml of 9% dilute sulfuric acid was added to the obtained “bacteria” and treated at 100 ° C. for 1 hour. Then, it neutralized with NaOH and centrifuged for 5 minutes at 15000 × g.
And 0.01 ml of obtained supernatants were taken and the amount of glucose was measured using glucose C-II test Wako (made by Wako Pure Chemical Industries, Ltd.). Among them, a strain that grew vigorously and accumulated a large amount of glucan (in this measurement, a strain with a high measured glucose level) was isolated and designated strain S24.

(2) Characteristics of bacterial cells (DNA sequence)
When the isolated strain S24 was observed with an electron microscope, it was 0.5-0.6 × 1-2 μm bacilli.
Next, genome extraction was performed from the cultured cells, and the base sequence encoding 16S rDNA was amplified by PCR using primers (27F [SEQ ID NO: 1] and 1522R [SEQ ID NO: 2]) with a thermal cycler. For this PCR product, four sequencing primers (S24-1 seq F01 [SEQ ID NO: 3], S24-1 seqF02 [SEQ ID NO: 4], S24-1 seqR01 [SEQ ID NO: 5], S24-1 seqR02 [SEQ ID NO: 6] As a result of analysis using a DNA sequencer, it was confirmed that the gene encoding 16S rDNA of the S24 strain consists of 1533 base pairs shown in SEQ ID NO: 7 below.
As a result of analyzing this sequence and the sequence obtained from Ribosomal Database Project II (http://rdp.cme.msu.edu/index.jsp) using the sequence analysis software GENETYX-WIN (Genetyx), The partial sequence [SEQ ID NO: 7] of 16S rDNA of S24 strain in the present invention showed 99.3% homology with the sequence of the standard strain of Enterobacter cancerogenus (LMG2693 strain).

When the bacteriological properties of this bacterium were examined in detail, the shape (gonococcus: 0.5 to 0.6 × 1 to 2 μm), Gram stain (−), Voges-Proskauer (VP) test (+), Indole Productivity (−), glucose fermentability (+), lactose degradability (+), urea degradability (−), DNase (−), nitrate reduction (+), citric acid availability (+), lysine decarbo Cyclase (-), ONPG (+), arginine dihydrolase (+), ornithine decarboxylase (+), hydrogen sulfide productivity (-), gelatin availability (-), glucose availability (+), D- Mannitol availability (+), inositol availability (-), D-sorbitol availability (+), L-rhamnose availability (+), sucrose availability (+), D-melibiose availability (+), L -It also shows properties such as arabinose use It was.
This revealed that the S24 strain in the present invention was a bacterium belonging to the genus Enterobacter, and was designated Enterobacter cancerogenus S24.
This strain has been deposited as “Enterobacter cancerogenus S24 (FERM P-21598)” at the Patent Organism Depository Center of the National Institute of Advanced Industrial Science and Technology (Tsukuba Center Central 1-1-1 Tsukuba City, Ibaraki Prefecture). ing.

<Example 2> Identification of type of glucan retained by S24 strain First, 20 ml of M9 liquid medium (composition is shown in Table 1) containing 1% arabinose was added to Enterobacter cancerogenus S24 strain (FERM P) obtained in Example 1. -21598), and precultured by shaking overnight at 30 degrees and 120 rpm.
10 ml of this preculture was inoculated into 100 ml of M9 medium containing 1% arabinose (into a 500 ml Erlenmeyer flask) and cultured at 30 ° C. and 120 rpm for 14 hours.
Then, after centrifuging the obtained microbial cell culture solution at 7000xg for 5 minutes and removing a culture medium, the precipitation part was wash | cleaned with water and centrifuged again, and the microbial cell which is a precipitation part was obtained.
Hydrolysis was performed by adding 5 ml of DMSO to this 'bacteria' and treating at 100 ° C for 1 hour. 40 ml ethanol was added to this solution, and the mixture was allowed to stand at 4 ° C. overnight, and then centrifuged at 10,000 × g for 30 minutes.
The obtained precipitate was washed once with 40 ml of ethanol and then centrifuged again to collect the precipitate, which was dried at 60 ° C. to obtain a “glucan-containing fraction”.

This 'glucan-containing fraction' is subjected to a degradation experiment using α-amylase (manufactured by Megazyme), pullulanase (manufactured by Hayashibara Biochemical Laboratories, Inc.), amyloglucosidase (manufactured by Megazyme), The degradation characteristics were examined and the type of glucan was identified.
First, 0.05 ml of 50 mM MOPS buffer (pH 7.0) and 15 U α-amylase were added to 0.21 mg of this “glucan-containing fraction” to make a total volume of 0.105 ml at 50 ° C. for 30 minutes. The reaction product was designated as reaction solution A ('α-amylase-treated section').
Further, 0.05 ml of this reaction solution A was separated, 0.05 ml of 50 mM sodium acetate buffer (pH 5.5) was added, and 1 U pullulanase was added to make a total volume of 0.105 ml, which was reacted at 50 ° C. for 30 minutes. Reaction solution B ('α-amylase + pullulanase treatment').
In addition, 0.05 ml of this reaction solution A was separated, 0.05 ml of 200 mM sodium acetate buffer (pH 4.5) was added, and 2 U of amyloglucosidase was added to make a total volume of 0.11 ml, which was reacted at 50 ° C. for 30 minutes. Was used as a reaction solution C ('α-amylase + amyloglucosidase treatment').
Further, 0.05 ml of this reaction solution A was separated, 0.05 ml of 200 mM sodium acetate buffer (pH 4.5), 15 U α-amylase, 2 U amyloglucosidase, and 1 U pullulanase were added to make a total amount of 0.115 ml. A reaction solution D ('α-amylase + amyloglucosidase + pullulanase treatment') was reacted at 50 ° C for 30 minutes.
Then, the amount of glucose in these reaction liquids A to D (the amount of glucose released by hydrolysis of the glucan) was quantified with a glucose C-II test Wako.

In addition, 0.1 ml of 9% dilute sulfuric acid was added to 0.5 mg of this “glucan-containing fraction” and treated at 100 ° C. for 1 hour to perform hydrolysis (“sulfuric acid treatment”). After neutralization, centrifugation was performed at 15000 × g for 5 minutes. Then, 0.01 ml of the obtained supernatant was taken, and the total amount of glucose released from glucan was measured using Glucose CII-Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.).
Then, the measured value of the amount of glucose released from each of the reaction solutions A to D is a relative value when the total glucose amount (quantified glucose-converted glucan amount) measured by the “dilute sulfuric acid treatment” is set to 100. Was calculated. The results are shown in FIG.

As a result, as shown in FIG. 1, the same amount as the “total glucose amount” was obtained by simultaneously treating with the above-described three kinds of enzymes (reaction solution D: “α-amylase + amyloglucosidase + pullulanase treatment”). It became clear that glucose was released.
In addition, the amount of glucose released was very small in the treatment with amylase alone (reaction solution A) or the treatment with a combination of amylase and pullulanase (reaction solution B). In addition, in the treatment with the combination of amylase and amyloglucosidase (reaction solution C), only about 70% of the “total glucose amount” released glucose.
From such enzymatic degradation characteristics, the glucan retained by the S24 strain was identified as 'glycogen' which is a kind of 'α-glucan'.

<Example 3> Retaining α-glucan of S24 strain First, 5 ml of M9 liquid medium (composition is shown in Table 1) containing 1% arabinose was inoculated with S24 strain (FERM P-21598) obtained in Example 1. Then, pre-culture was performed by culturing overnight at 30 degrees and 120 rpm.
This 'pre-culture solution' is centrifuged at 7000 × g for 5 minutes, and the cells recovered as a precipitate are washed twice with 10 mM phosphate buffer (pH 7.3) to obtain a “precipitate”. It was suspended in 5 ml of phosphate buffer.
1 ml of this “suspension” was inoculated into 10 ml of M9 medium containing 1% arabinose and cultured at 30 ° C. and 120 rpm for 14 hours.
Thereafter, 0.1 ml of the obtained “bacterial cell culture solution” was taken, centrifuged at 7000 × g for 5 minutes to remove the medium, and then the bacterial cell as a precipitation part was obtained.

Hydrolysis was carried out by adding 0.1 ml of 9% dilute sulfuric acid to the 'cells' and treating at 100 ° C for 1 hour. This was neutralized with an aqueous NaOH solution, and then centrifuged at 15000 × g for 5 minutes.
Then, 0.01 ml of the obtained supernatant was taken, and the amount of glucose was measured using Glucose CII-Test Wako (Wako Pure Chemical Industries, Ltd.). The amount of α-glucan retained in the cells was measured in terms of the amount of glucose (mg) detected from 1 ml of the cell culture solution. In addition, the retention rate of α-glucan was calculated. The results are shown in Table 2.

  As a result, it was found that “0.52 mg” of α-glucan in terms of glucose was obtained from 1 ml of the bacterial cell culture solution. Since the dry cell weight in 1 ml culture was 2.65 mg, it was revealed that the retention rate of α-glucan accounted for ‘19 .6% ’of the cell dry weight.

<Example 4> Utilization of carbohydrates of S24 strain First, instead of the M9 liquid medium containing 1% arabinose used in Example 3, each monosaccharide shown in Table 3 (L-arabinose, D-xylose, D-galactose, D-galacturonic acid, L-rhamnose, D-gluconic acid) was used in the same manner as in Example 3 except that an M9 liquid medium containing 1% as a carbon source was used. Pre-culture and culture (culture for 14 hours under the conditions of 30 ° C. and 120 rpm) were carried out to prepare a cell culture solution.
Thereafter, the obtained bacterial cell cultures were examined in the same manner as described in Example 3 by converting the amount of α-glucan retained per 1 ml of the bacterial cell cultures into the amount of glucose, and the bacterial cells. The retention of α-glucan per dry weight was examined. The results are shown in Table 3.

As a result, as shown in the table, the S24 strain assimilated L-arabinose, D-xylose, D-galactose, L-rhamnose, D-galacturonic acid, D-gluconic acid, and 10% of the dry weight of the cells. It was found that α-glucan accumulates in cells with a retention rate exceeding.
It was also found that the amount of glucan retention and glucan retention when L-arabinose was used as the carbon source was the highest.

<Example 5> Effect of concentration of monosaccharide substrate on α-glucan production First, S24 strain was pre-cultured in the same manner as described in Example 3 to 10 mM phosphate buffer (pH 7.3). A suspension was prepared. 1 ml of the solution was inoculated into 10 ml of M9 medium containing each concentration of arabinose described in Table 4. And it culture | cultivated for 14 hours on the conditions of 30 degreeC and 120 rotation / min.
Thereafter, for each of the bacterial cell culture solutions obtained, the amount of α-glucan retained per 1 ml of the bacterial cell culture solution was converted into the amount of glucose in the same manner as in Example 3, and the substrate was used. The 'conversion rate' in which a certain monosaccharide (arabinose) was converted to α-glucan (the amount of free glucose accumulated from α-glucan / the amount of arabinose in the culture solution before culture × 100) was calculated in terms of glucose. The results are shown in Table 4.

As a result, as shown in the table, when the concentration of arabinose as the substrate was 0.5%, the conversion rate of arabinose converted to α-glucan was '8.9%', and the concentration of arabinose as the substrate was 0. At 2%, the conversion rate was '22% '.
Therefore, in order to efficiently retain α-glucan in the cells under these culture conditions, the concentration of the monosaccharide as a substrate in the culture solution should be lower than 0.5%, particularly 0.2%. The following has proved effective.

<Example 6> Conversion of xylan hydrolyzate to α-glucan When hydrolyzate obtained by enzymatic treatment of xylan (Birchwood xylan, manufactured by Sigma), which is a kind of hemicellulose, is used as a substrate, to α-glucan by S24 strain The conversion and accumulation ability of was investigated.
First, in the same manner as described in Example 3, the S24 strain was pre-cultured and washed twice with 10 mM phosphate buffer (pH 7.3) to obtain a precipitate.
To this, 0.5 ml of “M9 medium containing“ 0.2% xylan ”and“ xylan-degrading enzyme (Bio-Feed Wheat L (0.47 mg / ml Novozymes) 0.01 ml) ”” was added and suspended. The cells were cultured at 40 ° C. and 120 rpm for 14 hours.
The precipitate was suspended in “0.5 ml of M9 medium containing 0.2% xylan” in “not containing xylan-degrading enzyme” and cultured in the same manner as a control.
Thereafter, in the same manner as the method described in Example 3, each obtained bacterial cell culture solution was examined by converting the amount of α-glucan retained per 1 ml of the bacterial cell culture solution into the amount of glucose. In addition, the retention rate of α-glucan per dry cell weight was examined. Further, according to the method described in Example 5, the “conversion rate” converted into α-glucan was calculated in terms of glucose. The results are shown in Table 5.

As a result, by adding “xylan-degrading enzyme” to the culture solution in addition to “0.2% xylan”, xylan is hydrolyzed, and this hydrolyzate is used as a substrate in the cells of S24 strain. It was shown that α-glucan is accumulated and retained. Moreover, it was shown that this reaction can be carried out in parallel side reactions.
The conversion rate of xylan (hydrolyzed xylan) in the medium into α-glucan was 12.5%.

<Example 7> Conversion of arabinoxylan hydrolyzate to α-glucan α-glucan produced by S24 strain using a hydrolyzate obtained by enzymatic treatment of arabinoxylan (manufactured by Megazyme), a kind of hemicellulose extracted from wheat. The ability to convert and accumulate was investigated.
First, in the same manner as described in Example 3, the S24 strain was pre-cultured and washed twice with 10 mM phosphate buffer (pH 7.3) to obtain a precipitate.
To this, “0.2% arabinoxylan” and “arabinoxylan degrading enzyme (Pectinex Ultra SPL (9500 U / ml, Novozymes) 0.01 ml, Celluclast 1.5 L (700 U / g, sigma) 0.01 ml, Viscozyme L (Batch KTN02157 , Novozymes) 0.01 ml) 'M9 medium "was added and suspended in 0.5 ml, and cultured at 40 ° C. under 120 rpm for 14 hours.
The precipitate was suspended in “without arabinoxylan” and suspended in “0.5 ml of M9 medium containing the arabinoxylan-degrading enzyme” and cultured in the same manner as a control.
Thereafter, in the same manner as the method described in Example 3, each obtained bacterial cell culture solution was examined by converting the amount of α-glucan retained per 1 ml of the bacterial cell culture solution into the amount of glucose. In addition, the retention rate of α-glucan per dry cell weight was examined. Further, according to the method described in Example 5, the “conversion rate” converted into α-glucan was calculated in terms of glucose. The results are shown in Table 6.

As a result, arabinoxylan is hydrolyzed by adding 'decomposition enzyme' to the culture medium in addition to 'arabinoxylan', and arabinoxylan is hydrolyzed, and α-glucan is formed in the cells of S24 strain using the hydrolyzate as a substrate. It was shown to be accumulated and retained. Moreover, it was shown that the reaction can be performed in parallel and multiple reactions.
Moreover, the conversion rate of arabinoxylan (hydrolysis product of arabinoxylan) in the medium into α-glucan was 23%.

<Example 8> Conversion of arabinan hydrolyzate to α-glucan The ability to convert and accumulate into α-glucan by S24 strain when a hydrolyzate of arabinan (manufactured by Megazyme), a kind of pectin, was used as a substrate. Examined.
First, a 2% aqueous sulfuric acid solution containing 2% arabinan (manufactured by Megazyme) was heat-treated at 100 ° C. for 1 hour, and then neutralized to pH 6.0 with CaCO ₃ . Then, after centrifuging at 10,000 × g for 10 minutes, the obtained supernatant was sterilized by filtration with a 0.2 μm filter to obtain a “solution containing arabinic acid-treated product (final sugar concentration 2%)”.
Next, in the same manner as described in Example 3, the S24 strain was precultured and washed twice with 10 mM phosphate buffer (pH 7.3) to obtain a precipitate.
To this, "M9 medium containing the above-mentioned 'arabinan hydrolyzate-containing solution (final sugar concentration 2%)' and 'pectin-degrading enzyme (Pectinex Ultra SP-L (9500 U / ml, Novozymes) 0.01 ml)" 0.5 ml was added and suspended, and cultured at 40 ° C. and 120 rpm for 14 hours.
In addition, “'(filter sterilized without acid treatment and 2% arabinan aqueous solution)” and “pectin-degrading enzyme (Pectinex Ultra SP-L (9500 U / ml, Novozymes) 0.01 ml)” are included. "M9 medium" 0.5 ml was added and suspended, and the same culture was used as a control.
Thereafter, in the same manner as the method described in Example 3, each obtained bacterial cell culture solution was examined by converting the amount of α-glucan retained per 1 ml of the bacterial cell culture solution into the amount of glucose. In addition, the retention rate of α-glucan per dry cell weight was examined. Further, according to the method described in Example 5, the “conversion rate” converted into α-glucan was calculated in terms of glucose. The results are shown in Table 7.

As a result, it was shown that in the case where “pectin hydrolase” was added to the culture solution in addition to “2% arabinan”, the arabinan was preferably treated with sulfuric acid in advance. By performing the flowing acid treatment, arabinan was significantly hydrolyzed, and α-glucan was accumulated and retained in the cells of the S24 strain using the hydrolyzate as a substrate.
The conversion rate of arabinan (arabinan hydrolyzate) into α-glucan in the medium was 20.5%.

<Example 9> Production of ethanol from low-utilization monosaccharide First, S24 strain (FERM P-21598) obtained in Example 1 was added to 100 ml of M9 liquid medium (composition is shown in Table 1) containing 1% arabinose. ) Was inoculated, and precultured by shaking culture overnight at 30 degrees and 120 rpm.
This preculture solution (total amount) was inoculated into 1000 ml of M9 medium containing 1% arabinose (into a 5000 ml Erlenmeyer flask) and cultured at 30 ° C. and 120 rpm for 14 hours.
Thereafter, in the same manner as in Example 3, the amount of α-glucan retained per 1 ml of the cell culture broth was converted into the amount of glucose, and the retention rate of α-glucan per cell dry weight was determined. It was.
The α-glucan retention rate of this microbial cell was “11.4%” per microbial cell dry weight.

Next, 200 ml of the obtained bacterial cell culture solution is centrifuged at 7000 × g for 5 minutes to remove the medium, and then the precipitate is washed with water and centrifuged again to obtain the bacterial cell that is the precipitate. It was.
Next, 5 ml of 50 mM MOPS buffer (pH 7.0) and 0.15 ml of α-amylase (manufactured by Megazyme) were added to the “bacteria” and reacted at 100 ° C. for 15 minutes. .
Next, 5 ml of 100 mM sodium acetate (pH 5.5), 0.15 ml of pullulanase (manufactured by Hayashibara Biochemical Laboratories, Inc.), and 0.15 ml of amyloglucosidase (manufactured by Megazyme) were added to this reaction solution. In addition, the mixture was reacted at 50 ° C. for 1 hour. The reaction solution was frozen and then concentrated under reduced pressure, and the total amount of the solution was 1 ml was designated as “enzyme reaction solution”.
The amount of glucose in this “enzyme reaction solution” (the amount of glucose liberated by hydrolysis of α-glucan accumulated in the cells) was quantified with a glucose C-II test Wako.

Next, this “enzyme reaction solution” was heated at 100 ° C. for 1 hour, and a fermentation experiment was performed using the yeast Saccharomyces cerevisiae.
First, yeast S. cerevisiae (NBRC224) pre-cultured overnight in YPD medium was centrifuged at 7000 × g for 5 minutes, washed twice with 10 mM phosphate buffer (pH 7.3), and the precipitate was removed. Obtained.
In this yeast precipitation part, 0.5 ml of the above-mentioned “enzyme reaction solution” was added and suspended so that the final OD (turbidity at 600 nm when using a cell with an optical path length of 1 cm) was 1.0, Ethanol fermentation was performed by culturing for 24 hours at 30 ° C. and 120 rpm.
In addition, the ethanol produced | generated in the obtained "ethanol fermentation liquid" was quantified by analyzing on the conditions shown in Table 8 using HPLC (LC Prominence, Shimadzu Corporation). In addition, the “conversion rate” in which the monosaccharide in the substrate medium was converted to ethanol was calculated. The results are shown in Table 9.

  As a result, it was shown that ethanol was produced by performing yeast fermentation using the 'enzyme reaction solution' of S24 strain obtained by the above method as a raw material. Further, it was shown that 74% of glucose contained in the “enzyme reaction solution” (the α-glucan was enzymatically decomposed) was converted to ethanol.

[Arthrobacter sp. 4P-01-A1 (FERM P-21807) strain]
<Example 10> Selection and identification of 4P-01-A1 strain (1) Selection of strains In addition to the above strains, microorganisms having the ability to assimilate arabinose and accumulate in large quantities as α-glucans were selected. .
First, 0.1 mg of soil sample was suspended in 1 ml of sterilized water, diluted appropriately and 0.2% YP (0.4% Tryptone, 0.2% Yeast extract, pH 6.8) 2% agar medium containing 0.1% arabinose. And cultured at 30 ° C. overnight. Subsequently, the colonies obtained on the agar medium after the culture were picked up with a toothpick, and using a 96-well plate, 0.2 × YP liquid medium (0.4% Tryptone, 0.2% containing 0.6 ml of 1% arabinose) was used. % Yeast extract, pH 6.8), and cultured for 24 hours at 30 ° C. and 120 rpm.
Thereafter, 0.1 ml of the obtained “bacterial cell culture solution” was taken and centrifuged at 7000 × g for 5 minutes to remove the medium, and then the bacterial cell as a precipitation part was obtained.
0.05 ml of BugBuster protein extraction reagent (Novagen) was added to the obtained “bacteria” and treated at 30 ° C. for 15 minutes to perform cell degradation. Then 0.05 ml of enzyme solution “50 μM sodium acetate buffer (pH 5.0) containing“ 0.25 μl Liquozyme SC (Amylase) (Novozyme) ”and“ 0.25 μl Spirizyme Fuel (AMG) (Novozyme) ”was added, and 50 Treated for 1 hour at ° C. After the enzyme reaction, the mixture was centrifuged at 5000 × g for 10 minutes, 10 μl of the supernatant fraction was taken, the amount of glucose was measured, and bacteria that accumulate a large amount of α-glucan were selected. Among them, a strain that accumulates a large amount of α-glucan was isolated and designated 4P-01-A1 strain.

(2) Characteristics of bacterial cells (DNA sequence)
The isolated strain 4P-01-A1 was subjected to genome extraction from the cultured cells, and a base sequence encoding 16S rDNA was obtained using primers (27F [SEQ ID NO: 1] and 1522R [SEQ ID NO: 2]) with a thermal cycler. Amplification was performed by PCR, and the sequence was analyzed.
Then, Example 1 was used except that the following four primers (27F [SEQ ID NO: 1], 1522R [SEQ ID NO: 2], 800R [SEQ ID NO: 8], 800R-F [SEQ ID NO: 9]) were used. Similarly, as a result of analysis using a DNA sequencer, it was confirmed that the gene encoding 16S rDNA of the 4P-01-A1 strain consists of 1472 base pairs shown in SEQ ID NO: 10 below.
As a result of comparative analysis of this sequence and the sequence in the database of DDBJ-16S rRNA by BLAST search, a partial sequence [SEQ ID NO: 10] of 4P-01-A1 strain in the present invention was found to be the Arthrobacter nitroguaiacolicus (AJ512504). It showed 99% and 2% homology with the sequence of the standard strain (CCM4924 strain).
This revealed that the 4P-01-A1 strain in the present invention is a bacterium belonging to the genus Arthrobacter.
In addition, this strain is registered in the Patent Organism Depositary Center of the National Institute of Advanced Industrial Science and Technology (Tsukuba Center Central 1-1-1 Tsukuba City, Ibaraki Prefecture) “Arthrobacter sp. 4P-01-A1 (FERM P-21807 ) ".

<Example 11> Identification of type of glucan retained by 4P-01-A1 strain Obtained in Example 10 in 5 ml of 0.2 × YP medium (0.4% Tryptone, 0.2% Yeast extract) containing 1% arabinose 4P-01-A1 strain (FERM P-21807) was inoculated and precultured by shaking overnight at 30 ° C. and 250 rpm.
The obtained preculture was washed with 10 mM phosphate buffer (pH 7.3), 0.2 × YP medium (0.4% Tryptone, 0.2% Yeast extract) containing 1% arabinose, 100 ml at 30 ° C., 250 ° The cells were cultured for 24 hours at a rotation / minute to prepare a cell culture solution.
30 ml of the obtained bacterial cell culture solution was collected by centrifugation and treated with 1 ml of 50 mM MOPS (pH 7.0) at 100 ° C. for 10 minutes to lyse the cells. Then, after centrifugation at 20000 × g for 5 minutes, the supernatant was collected as a glucan-containing fraction. For this glucan-containing fraction, the type of glucan was identified in the same manner as in the method described in Example 2. The results are shown in FIG.

As shown in the figure, in the treatment with amylase alone (reaction solution A), the amount of released glucose was extremely small. In addition, in the treatment with the combination of amylase and pullulanase (reaction solution B), about 70% of the “total glucose amount”, and in the treatment with the combination of amylase and amyloglucosidase (reaction solution C), about “total glucose amount”. Only 40% of glucose was released.
On the other hand, it is clear that the same amount of glucose as 'total glucose amount' is released by simultaneous treatment with three kinds of enzymes (reaction solution D: 'α-amylase + amyloglucosidase + pullulanase treatment'). became.
From such enzymatic degradation characteristics, the glucan retained by the 4P-01-A1 strain was identified as “glycogen” which is a kind of “α-glucan”.

<Example 12> α-glucan retention of 4P-01-A1 strain Obtained in Example 10 in 5 ml of 0.2 × YP liquid medium (0.4% Tryptone, 0.2% Yeast extract, pH 6.8) containing 1% arabinose. 4P-01-A1 strain (FERM P-21807) was inoculated and precultured by shaking overnight at 30 ° C. and 250 rpm.
0.2 ml of this preculture was inoculated into 10 ml of YP liquid medium containing 1% arabinose, and cultured for 24 hours at 30 ° C. and 250 rpm.
Thereafter, the obtained bacterial cell culture solution was examined in the same manner as in Example 3 by converting the amount of α-glucan retained per 1 ml of the bacterial cell culture solution into the amount of glucose. In addition, the retention rate of α-glucan per dry cell weight was examined. The results are shown in Table 10.

  As a result, it was found that “0.49 mg” of α-glucan was obtained in terms of glucose from 1 ml of the bacterial cell culture solution. And it became clear that the retention rate of α-glucan accounted for ‘19 .5% ’of the dry weight of the cells.

<Example 13> 4P-01-A1 strain carbohydrate assimilation 4P obtained in Example 10 in 5 ml of 0.2 × YP medium (0.4% Tryptone, 0.2% Yeast extract) containing 1% arabinose -01-A1 strain (FERM P-21807) was inoculated and precultured by shaking culture overnight at 30 ° C and 250 rpm.
This preculture was washed with 10 mM phosphate buffer (pH 7.3), then washed with 10 ml of a medium containing 0.4% Tryptone, 0.2% Yeast extract, 1% of various carbohydrates at 30 ° C., 250 rpm for 24 rpm. Time culture was performed.
Thereafter, the obtained bacterial cell culture solution was examined in the same manner as in Example 3 by converting the amount of α-glucan retained per 1 ml of the bacterial cell culture solution into the amount of glucose. In addition, the retention rate of α-glucan per dry cell weight was examined. The results are shown in Table 11.

As a result, as shown in the table, 4P-01-A1 strain assimilated cellobiose, L-arabinose, D-xylose, D-galacturonic acid, D-gluconic acid, L-rhamnose, and dried the cells. It was found that α-glucan was accumulated in the cells with a retention rate of 13 to 24.8% by weight.
It was also found that the glucan retention rate was highest when L-arabinose was used as the carbon source.

[About Sporosarcina sp. N52 (FERM P-21808) strain]
<Example 14> Selection and identification of N52 strain (1) Selection of strains In addition to the above strains, microorganisms having the ability to assimilate arabinose and accumulate in large quantities as α-glucans were selected.
First, 0.1 mg of a soil sample is suspended in 1 ml of sterilized water, diluted appropriately, and spread on a 2% agar medium of YP (2% Tryptone, 1% Yeast extract) containing 0.1% arabinose at 30 ° C. Cultured overnight. Next, the colonies obtained on the agar medium after the cultivation were picked with a toothpick and inoculated into a YP liquid medium (2% Tryptone, 1% Yeast extract) containing 0.1% arabinose, and rotated at 30 ° C. and 120 rpm. The culture was performed for 24 hours under the conditions of / min.
Thereafter, 0.1 ml of the obtained “bacterial cell culture solution” was taken and centrifuged at 7000 × g for 5 minutes to remove the medium, and then the bacterial cell as a precipitation part was obtained.
To the obtained 'cells', 0.1 ml of 9% dilute sulfuric acid was added and treated at 100 ° C for 1 hour. Thereafter, the mixture was neutralized with NaOH and centrifuged at 15000 × g for 5 minutes.
0.01 ml of the obtained supernatant was taken and the amount of glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.). Among them, a strain that grew quickly and accumulated glucan (a strain having a large amount of glucose in this measurement) was isolated and designated as N52 strain.

(2) Characteristics of bacterial cells (DNA sequence)
The isolated strain N52 is subjected to genome extraction from the cultured cells, and the PCR is used to amplify the base sequence encoding 16S rDNA using primers (27F [SEQ ID NO: 1] and 1522R [SEQ ID NO: 2]) with a thermal cycler. The sequence was analyzed.
Then, the following six primers (27F [SEQ ID NO: 1], 1522R [SEQ ID NO: 2], N52-F01 [SEQ ID NO: 11], N52-F02 [SEQ ID NO: 12], N52-R01 [SEQ ID NO: 13], N52 As a result of analysis using a DNA sequencer in the same manner as in Example 1 except that -R02 [SEQ ID NO: 14] was used, the gene encoding 16S rDNA of the N52 strain was 1544 base pairs shown in SEQ ID NO: 15 below. It was confirmed to consist of
As a result of comparative analysis of this sequence and the sequence in the database of DDBJ-16S rRNA by BLAST search, the partial sequence [SEQ ID NO: 15] of N52 strain in the present invention is a standard strain of Sporosarcina ginsengisoli (Gsoil 1433 strain). And 99.7% homology with the sequence.
This revealed that the N52 strain in the present invention is a bacterium belonging to the genus Sporosarcina and was designated Sporosarcina sp. N52.
The strain is deposited as “Sporosarcina sp. N52 (FERM P-21808)” at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (Tsukuba Center Central 1-1-1 Tsukuba City, Ibaraki Prefecture). Has been.

<Example 15> Identification of the type of glucan retained by the N52 strain In 5 ml of 0.2 × YP medium (0.4% Tryptone, 0.2% Yeast extract) containing 1% arabinose, the N52 strain (P -21808) was inoculated, and precultured by shaking overnight at 30 ° C. and 250 rpm.
This preculture was washed with 10 mM phosphate buffer (pH 7.3) and then cultured in 100 ml of a medium consisting of 2% Tryptone, 1% Yeast extract, 1% arabinose at 30 ° C., 250 rpm for 24 hours. .
35 ml of the obtained bacterial cell culture solution was collected by centrifugation and treated with 1 ml of 50 mM MOPS (pH 7.0) at 100 ° C. for 10 minutes to decompose the cells. Then, after centrifugation at 20000 × g for 5 minutes, the supernatant was collected as a glucan-containing fraction. The kind of glucan was identified in the same manner as in Example 2 for this glucan-containing fraction. The results are shown in FIG.

As shown in the figure, in the treatment with amylase alone (reaction solution A), the amount of released glucose was extremely small. In addition, in the treatment with the combination of amylase and pullulanase (reaction solution B), about 70% of the “total glucose amount”, and in the treatment with the combination of amylase and amyloglucosidase (reaction solution C), about “total glucose amount”. Only 20% of the glucose was released.
On the other hand, it is clear that the same amount of glucose as 'total glucose amount' is released by simultaneous treatment with three kinds of enzymes (reaction solution D: 'α-amylase + amyloglucosidase + pullulanase treatment'). Became.
From such enzymatic degradation characteristics, the glucan retained by the N52 strain was identified as “glycogen” which is a kind of “α-glucan”.

<Example 16> α-glucan retention of N52 strain 5 ml of YP liquid medium (2% Tryptone, 1% Yeast extract) containing 1% arabinose was inoculated with the N52 strain (P-21808) obtained in Example 14. Pre-culture was performed by shaking culture overnight at 30 ° C. and 250 rpm.
0.2 ml of this preculture was inoculated into 10 ml of YP liquid medium containing 1% arabinose, and cultured for 24 hours at 30 ° C. and 250 rpm.
Thereafter, the obtained bacterial cell culture solution was examined in the same manner as in Example 3 by converting the amount of α-glucan retained per 1 ml of the bacterial cell culture solution into the amount of glucose. In addition, the retention rate of α-glucan per dry cell weight was examined. The results are shown in Table 12.

  As a result, it was found that 2.39 mg of α-glucan was obtained in terms of glucose from 1 ml of the cell culture solution. Since the dry cell weight in 1 ml culture was 8.58 mg, it was revealed that the retention rate of α-glucan accounted for 27.9% of the dry cell weight.

<Example 17> Glucose assimilation of N52 strain The N52 strain (P-) obtained in Example 14 was added to 5 ml of 0.2 × YP medium (0.4% Tryptone, 0.2% Yeast extract) containing 1% arabinose. 21808) was inoculated and precultured by shaking overnight at 30 ° C. and 250 rpm.
This preculture was washed with 10 mM phosphate buffer (pH 7.3), and then 10 ml of a medium containing 0.4% Tryptone, 0.2% Yeast extract, and 1% of various carbohydrates at 30 ° C., 250 rpm / Incubated for 24 hours in minutes.
Thereafter, the obtained bacterial cell culture solution was examined in the same manner as in Example 3 by converting the amount of α-glucan retained per 1 ml of the bacterial cell culture solution into the amount of glucose. In addition, the retention rate of α-glucan per dry cell weight was examined. The results are shown in Table 13.

As a result, as shown in the table, the N52 strain assimilated cellobiose, L-arabinose, D-xylose, D-galacturonic acid, D-gluconic acid, L-rhamnose, It was found that α-glucan was accumulated in the cells with a retention rate of 36%.
It was also found that the glucan retention rate was particularly high when L-arabinose, D-xylose, and D-gluconic acid were used as the carbon source.

<Example 18> Treatment of sweet potato distillation residue by N52 strain Using the residue after ethanol fermentation of sweet potato, it was converted into α-glucan.
After pulverizing 3 kg of sweet potato (variety: Daiichinoume), liquefying it with a jet cooker (Noritake Engineering Co., Ltd. (NCP-250 / 20-3 / 3)), using a jar fermenter (5L), 30 ° C, pH 5 The yeast S. cerevisiae was subjected to ethanol fermentation using the strain (NBRC224) for 5 days under the conditions described above. In addition, the composition optimized in potato in Srichuwong S. et al., 2009, Biomass and Bioenrgy, 33: 890-898.
Thereafter, the entire mash obtained was distilled at 99 ° C. using a solvent regenerator (SR-2000) manufactured by EEYLA to make the ethanol concentration of the distillation residue 0.18%. This distillation residue (50 ml) was centrifuged at 15000 rpm for 10 minutes, and then the supernatant was passed through a 0.2 μm filter to sterilize to obtain a “distillation waste liquid”.

  Next, the N52 strain obtained in Example 14 was inoculated into 10 ml of 0.2 × YP medium (0.4% Tryptone, 0.2% Yeast extract) containing 1% arabinose, and the conditions were 30 ° C. and 250 rpm. Pre-culture was performed by overnight shaking culture. Thereafter, the cells are washed with 10 mM phosphate buffer (pH 7.3), and the obtained washed cells are suspended in a small amount of sterilized water, and the cell suspension with a turbidity (OD600) value of 120 is used. 'I got.

And after transferring 50 ml of the above-mentioned distillation waste liquid derived from sweet potato to a 200 ml Erlenmeyer flask, the cell suspension of N52 was added to inoculate the microbial cell suspension so that OD600 would be 1 after addition, and 30 ° C., 250 ° C. Incubate for 72 hours at rev / min.
In addition, it sampled in the middle of culture | cultivation, and the microbial cell dry weight and the amount of (alpha)-glucan accumulated in the microbial cell were measured similarly to the method as described in Example 3.
Moreover, about the quantity of main monosaccharide and disaccharide contained in a culture solution, ion chromatography [Dionex ICS-3000 (Dionex Corp., Sunnyvale, CA), column: Carbo-Pac PA1, column temperature: 20 degree | times] , Detector: pulsed amperometric detector, flow rate: 1 ml / min.]. Monosaccharides were separated with water for 30 minutes, and disaccharides and GalA were separated with 150 mM NaOH and 150 mM NaOH-185 mM sodium acetate for 15 minutes. The column was washed with 150 mM NaOH-500 mM sodium acetate for 15 minutes, and the column was regenerated with 150 mM NaOH for 12 minutes.
FIG. 4 shows the measurement results of “bacterial cell dry weight” and “α-glucan retention amount”.
Moreover, the measurement result of the quantity of main residual monosaccharide and disaccharide contained in the distillation waste liquid (culture liquid) is shown in FIG. (In FIG. 5, “GalA” is galacturonic acid, “Cellobiose” is cellobiose, “Gal” is galactose, “Ara” is arabinose, “Glc” is glucose, “Rha” is rhamnose, “Xyl” is xylose, "Fructose" indicates fructose and "Man" indicates mannose.)
Table 14 shows the results of summarizing numerical data relating to the production process of glucan from sweet potato waste liquor.

As a result, as shown in FIG. 4, the dry weight of bacterial cells and the amount of retained glucan in the cells increased with the passage of the culture time, and reached the maximum after 48 hours of culture. Further, as shown in FIG. 5, it was clarified that the main remaining monosaccharide and disaccharide contained in the waste liquid derived from sweet potato were completely assimilated by 48 hours of cultivation.
From these results, in the production process of glucan from sweet potato waste liquor, the main residual monosaccharide and disaccharide (9.3 mg / ml) contained in the distillate waste are assimilated by the N52 strain, and 4.3 mg / ml. It was shown that ml glucan was produced. Moreover, it was found that the glucan retention rate of the bacterial cells was 32%.

The present invention relates to a technology for effectively using resources by-produced in the biological industry. By converting resources such as carbohydrates, which have low utility, especially plant cell wall components, to α-glucan that can be used as an energy source by many organisms, not only can resources be used effectively, but food, feed, It can provide new possibilities in the fermentation industry.
Furthermore, the present invention promotes metabolic studies for the purpose of elucidating the accumulation mechanism of α-glucan, and accelerates molecular breeding studies utilizing microbial breeding and genetic recombination techniques, thereby improving α-glucan productivity. Improvement is realized.
In addition, in the waste liquid treatment step, it is possible to obtain glucan-carrying cells while reducing BOD.

FERM P-21598
FERM P-21807
FERM P-21808

Claims (17)

  The plant cell wall component is hydrolyzed to a monosaccharide by treatment with an acid or an enzyme that hydrolyzes the plant cell wall component; and α-glucan is obtained by assimilating the monosaccharide in a culture solution containing the monosaccharide. By culturing bacteria belonging to the Proteobacteria class, Actinobacteria class, or Bacilli class, which have the property of being converted to and retaining them, and assimilating the monosaccharides; A method for producing a bacterial cell that retains α-glucan converted from a plant cell wall component, wherein glucan is accumulated in the bacterial cell.   The monosaccharide is at least one of D-xylose, L-arabinose, D-galacturonic acid, L-rhamnose, D-glucuronic acid, D-galactose, and D-mannose. The manufacturing method of the microbial cell holding the alpha-glucan of Claim 1.   The microbial cell holding the α-glucan according to claim 1, wherein the monosaccharide in the culture medium is converted into α-glucan in an amount of 10% or more of the amount of the monosaccharide in terms of glucose. Manufacturing method.   The α- of any one of claims 1 to 3, wherein the concentration of the monosaccharide in the culture solution is 0.05% (w / v) or more and less than 0.5% (w / v). A method for producing bacterial cells that retain glucan.   A plant cell wall component is hydrolyzed by treatment with an acid or an enzyme that hydrolyzes the plant cell wall component, and bacteria belonging to the Proteobacteria class, Actinobacteria class, or Bacilli class are cultured in a culture solution containing the hydrolyzate. Then, by assimilating the hydrolyzate, from the plant cell wall component characterized by accumulating 10% or more of α-glucan per dry weight of the bacteria in the bacteria A method for producing a microbial cell retaining a converted α-glucan.   When the bacteria are selected from the soil, they are selected using as an index the property of assimilating the monosaccharide to accumulate 10% or more α-glucan in the bacterial cells by dry weight of the bacteria. The manufacturing method of the microbial cell which hold | maintains the alpha-glucan in any one of Claims 1-5.   The manufacturing method of the microbial cell holding the alpha-glucan in any one of Claims 1-6 whose said bacteria are bacteria which belong to Enterobacter genus, Arthrobacter genus, or Sporosarcina genus.   The bacterium is Enterobacter cancerogenus S24 (FERM P-21598), Arthrobacter sp. 4P-01-A1 (FERM P-21807), or Sporosarcina sp. N52 (FERM P-21808), or a mutant thereof. The manufacturing method of the microbial cell holding the alpha-glucan in any one of Claims 1-7.   The manufacturing method of the microbial cell which hold | maintains the alpha-glucan in any one of Claims 1-8 whose said plant cell wall component has hemicellulose and / or pectin as a main component.   The manufacturing method of the microbial cell holding the alpha-glucan in any one of Claims 1-9 whose said plant cell wall component is what has xylan as a main component.   The manufacturing method of the microbial cell holding the alpha-glucan in any one of Claims 1-10 whose said alpha-glucan is what has glycogen as a main component.   The method for producing a microbial cell retaining α-glucan according to any one of claims 1 to 11, wherein the bacterium is cultured and then solid-liquid separated to recover the microbial cell as a solid content.   A microbial cell retaining α-glucan obtained by the production method according to claim 1.   After destroying the microbial cell holding the α-glucan according to claim 13, the α-glucan is treated with at least one enzyme of α-amylase, β-amylase, pullulanase, amyloglucosidase, and α-glucosidase. To produce glucose by hydrolysis.   Glucan is hydrolyzed to glucose by treating the bacterial cells holding α-glucan according to claim 13 at a temperature of 60 ° C or higher using dilute hydrochloric acid or dilute sulfuric acid. Manufacturing method.   A method for producing ethanol, characterized in that glucose obtained by the production method according to claim 14 or 15 is used as a raw material for ethanol fermentation.   Food, feed or food containing α-glucan contained in a microbial cell retaining α-glucan according to claim 13, or glucose obtained by the production method according to claim 14 or 15. Microbial nutrient source.

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