JP2012005359A - Method for manufacturing monosaccharide, disaccharide, and/or oligosaccharide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently manufacturing a degraded saccharide from a polysaccharide by improving a saccharization rate.SOLUTION: The method for manufacturing a monosaccharide, a disaccharide, and/or an oligosaccharide includes: a step (1) in which a polysaccharide is pulverized via the application of compression shear stress, yielding a pulverized polysaccharide; a step (2) in which the pulverized polysaccharide is heat-treated at 185-230°C in the presence of water; and a step (3) in which the solid portion of the heat-treated reactant is obtained via solid-liquid separation and acted on by a hydrolase.

Description

  The present invention relates to a method for producing polysaccharides, particularly monosaccharides, disaccharides, and / or oligosaccharides (hereinafter collectively referred to as “decomposed sugars”) from biomass.

In recent years, development of technology for producing useful substances such as ethanol and lactic acid from cellulose-based, starch-based, or saccharide-based biomass in place of fossil resources has been desired. In particular, there is a growing interest in technologies that make effective use of cellulosic biomass, which is a non-food resource.
Production of ethanol or the like from biomass can be performed by decomposing biomass into sugars such as glucose in the saccharification step, and then converting this to ethanol or the like in the fermentation step. Saccharification is roughly divided into acid saccharification using sulfuric acid or the like and enzyme saccharification.
Enzymatic saccharification has the advantage that it can be hydrolyzed under mild conditions compared to acid saccharification, but especially when cellulosic biomass is used, the reaction rate is very slow and increasing the saccharification rate is a major issue. Yes.

As a technique for improving the saccharification rate, a method of pretreating biomass in advance prior to the saccharification step has been studied. As the pretreatment method, for example, a sulfuric acid method, an organic solvent method, a hydrothermal treatment method, mechanical crushing, and the like are known.
Also, a method for efficiently producing an ethanol raw material by combining these methods has been studied. For example, a coarse pulverization step for coarsely pulverizing a raw material containing lignocellulose, a fine pulverization step for finely pulverizing a coarsely pulverized product, and fine pulverization A method (Patent Document 1) including a hydrothermal treatment step of hydrothermally treating an object and a dehydration step of dehydrating the hydrothermal treatment product has been reported.

JP 2009-124973 A

Patent Document 1 describes that when the finely pulverized product obtained in the pulverization step is hydrothermally treated, about 0.98 MPa (about 180 ° C. or less as saturated steam) is preferable in order to suppress excessive decomposition of sugar. is there. However, the Patent Document 1 does not disclose the implementation of hydrothermal treatment, and the present inventor performed the pulverization step of cellulose-based biomass and the hydrothermal treatment step according to the method described in Patent Literature 1, and then the actual enzymatic saccharification step. However, although the yield of cellulose after the hydrothermal treatment step is high, it has been found that the reaction is difficult to proceed in the enzymatic saccharification step, and the amount of decomposition sugar produced as a whole may be reduced. On the other hand, even if cellulosic biomass is simply mechanically crushed and then subjected to enzymatic saccharification, the reaction rate decreases with time, and a sufficient saccharification rate may not be obtained.
Therefore, the subject of this invention is improving the saccharification rate and providing the method which can manufacture a decomposition sugar from a polysaccharide efficiently.

  The inventors of the present invention have intensively studied in view of the above problems. As a result, when the polysaccharide is pulverized by a specific method and then heat-treated at 185 to 230 ° C. in the presence of water, the temperature is low as in Patent Document 1. Although the yield of cellulose after heat treatment is lower than when heat-treated, the subsequent enzymatic saccharification step unexpectedly improves the saccharification rate significantly and ultimately increases the yield of decomposed sugar. I found it.

That is, the present invention includes the following steps (1) to (3):
(1) A step of applying a compressive shear stress to a polysaccharide to pulverize the polysaccharide to obtain a pulverized polysaccharide;
(2) A step of heat-treating the pulverized polysaccharide at 185 to 230 ° C. in the presence of water,
(3) A step of obtaining a solid part by solid-liquid separation from the reaction product after the heat treatment, and allowing a hydrolase to act on the solid part,
A method for producing monosaccharides, disaccharides, and / or oligosaccharides is provided.

  According to the present invention, the saccharification rate can be improved, and a decomposing sugar can be produced efficiently. The method of the present invention can also be applied to cellulosic biomass, and can be expected as a technique for improving the production efficiency of useful substances such as ethanol from biomass.

Step (1) is a step of obtaining a pulverized polysaccharide by pulverizing the polysaccharide by applying compressive shear stress.
Examples of the polysaccharide used in the present invention include cellulose, hemicellulose, xyloglucan, pectin, starch, mannan, glucomannan, galactomannan, chitin, chitosan, inulin, alginic acid, agar, fucoidan, laminarin, β-glucan, pullulan. Natural polysaccharides such as these or derivatives thereof. These can be used alone or in combination of two or more. Of these, cellulose, hemicellulose, chitin, and chitosan are preferable, and cellulose and hemicellulose are particularly preferable because they are inexpensive and can be converted into useful substances by fermentation production after decomposition. The molecular weight of the polysaccharide used in the present invention is not particularly limited, but generally it is preferably 1,000 or more and 5,000,000 or less.

Moreover, the raw material containing the said polysaccharide, for example, biomass, can also be used as a polysaccharide in this invention. Biomass is an organic resource derived from living organisms, excluding fossil resources. Examples of the biomass include cellulose-based, starch-based, and saccharide-based biomass, and these can be used alone or in combination of two or more.
Especially, it is preferable to use the cellulose biomass containing a cellulose from the point of effective utilization of resources.

  Cellulosic biomass is mainly composed of cellulose, hemicellulose, and lignin. For example, cotton, wood pulp, kenaf, hemp, small-diameter tree, thinned wood, sawdust, wood chips, waste paper, newspaper, wrapping paper, tissue paper Woody materials such as toilet paper and cardboard; plant biomass such as bagasse, switchgrass, elephant grass, rice straw and wheat straw. Examples of the starch-based biomass include rice, wheat, corn, and potato, and examples of the saccharide-based biomass include sugar-based biomass such as sugar cane, sugar beet, seaweed, shrimp shell, and crab shell.

In order to pulverize the polysaccharide by applying a compression shear stress, a compression shearing pulverizer can be used. The compression shearing type pulverizer is a machine that can apply both compressive stress and shear stress, and examples thereof include a vibrating rod mill and a vibrating ball mill. Among these, a vibrating rod mill is preferable from the viewpoint of saccharification rate and production efficiency. The rod is not particularly limited, but preferably has an outer diameter of 0.1 to 100 mm, and more preferably 0.5 to 50 mm. Moreover, although the filling rate (the apparent volume of the rod with respect to the volume of the stirring part of the vibration mill) varies depending on the model, it is preferably 10 to 97%, more preferably 15 to 95%.
The pulverization conditions such as the pulverization time and the rotational speed of the pulverizer may be set as appropriate in order to form a desired pulverized product.

  The average particle size of the pulverized product is preferably 0.002 to 0.3 mm, more preferably 0.005 to 0.2 mm, and particularly preferably 0.01 to 0.05 mm from the viewpoint of saccharification rate and production efficiency. In addition, in this specification, an average particle diameter means the average value calculated | required according to a volume reference | standard by the laser diffraction scattering method.

In addition, the polysaccharide may be coarsely pulverized in advance before being pulverized by applying compressive shear stress. The method for coarse pulverization is not particularly limited, and for example, as a pulverizer, a cutter pulverizer such as a grinder or roll cutter, an impact pulverizer such as a hammer mill, a grinding pulverizer such as a colloid mill, or the like may be used. it can.
The coarse pulverization is preferably performed so that the average particle size of the polysaccharide is 1 to 15 cm, particularly 3 to 5 cm.

Step (2) is a step of heat-treating the pulverized product obtained in step (1) at 185 to 230 ° C. in the presence of water. It does not restrict | limit especially as a heat processing method, A well-known method is applicable. Batch stirring type or continuous flow type may be used. Examples of water used in the heat treatment include tap water, distilled water, ion exchange water, purified water, and the like.
The pulverized product is preferably used in the form of a slurry, and the content of the polysaccharide in the slurry is 1 to 400 g / L, more preferably 5 to 300 g / L, particularly 8 to 200 g / L from the viewpoint of fluidity. Is preferred. Examples of the slurry include water and various buffer solutions.

  Although the temperature of heat processing is the range of 185 degreeC-230 degreeC, it is more preferable from the point of saccharification rate and production efficiency that it is 185-220 degreeC, especially 190-210 degreeC. Examples of the heating means include water vapor and electricity.

  The time for heat treatment in the range of 185 ° C. to 230 ° C. varies depending on the reaction method and the type of polysaccharide, but is preferably 10 to 90 minutes, more preferably 15 to 85 minutes, particularly 20 to 20 minutes from the viewpoint of the decomposition rate and production efficiency. 80 minutes is preferred. Note that the heat treatment time means the total time in a state included in the specified temperature range, and the temperature may change as long as the temperature is included in the range. Accordingly, the temperature raising and lowering times are included in the heat treatment time as long as they are included in the temperature range. Further, when the temperature is intermittently included in the temperature range as a result of the temperature change, the total time included in the temperature range is set as the time.

  The pressure during the heat treatment is preferably set to the saturated vapor pressure of water or higher, more preferably 1.1 to 10.0 MPa, particularly 1.2 to 8.0 MPa, and particularly 1.3 to 6.0 MPa. Is preferred. In the case of pressurization, a gas may be used, and examples of the gas used include inert gas, water vapor, nitrogen gas, and helium gas. In the case of a continuous flow type, the pressure may be set by restricting the outlet channel with a valve or the like.

Step (3) is a step of obtaining a solid part by solid-liquid separation from the reaction product after the heat treatment, and allowing a hydrolase to act on the solid part. By removing the liquid part from the reaction product, it is possible to remove the overlysate, and to prevent fermentation inhibition by the sugar overlysate in the subsequent fermentation step.
The method for obtaining the solid part from the reaction product after the heat treatment by solid-liquid separation is not particularly limited, and can be performed by filtration, centrifugation, and sedimentation while cooling as necessary. The cooling temperature is 100 ° C. or lower, preferably 80 ° C. or lower.

The hydrolase used in the present invention is not particularly limited as long as it has an activity of hydrolyzing polysaccharides. For example, amylase, glucoamylase, cellulase, dextranase, glucanase, glucosidase, galactosidase, Mannosidase, agarase, lactose, mutanase, chitinase, chitosanase and the like can be mentioned.
The origin of the hydrolase is not limited, and it may be an artificial enzyme by gene recombination technique, partial hydrolysis or the like.
The form of the hydrolase is not particularly limited, and dried enzyme protein, particles containing enzyme protein, liquid containing enzyme protein, and the like can be used.

  The amount of hydrolase used varies depending on the conditions of the hydrolysis reaction, the type of polysaccharide, and the like. For example, in the case of cellulase, 1 to 200 FPU is preferable per 1 g of the solid part, and 5 to 50 FPU is particularly preferable. Here, the FPU activity is measured by the IUPAC method shown below. First, 50 mg of filter paper (Whatman No. 1) is used as a substrate, 0.5 mL of enzyme solution and 1.0 mL of 0.05 M citrate buffer (pH 4.8) are added thereto, and the enzyme reaction is carried out at 50 ° C. for 1.0 hour. Then, 3.0 mL of dinitrosalicylic acid reagent is added, and the mixture is heated at 100 ° C. for 5.0 minutes for color development. After cooling, 20 mL of ion-exchanged water is added thereto, and colorimetric determination is performed at a wavelength of 540 nm. The amount of enzyme that produces a reducing sugar corresponding to 1 μmol of glucose per minute is defined as 1 unit (FPU).

  For the reaction between the polysaccharide and the hydrolase, the temperature and pH can be selected according to the characteristics of the enzyme used. For example, when cellulase is used, the pH should be 3 to 8, particularly pH 4 to 7. The temperature is preferably 10 to 80 ° C, particularly preferably 20 to 60 ° C.

  Although reaction time changes with kinds etc. of polysaccharide, from the point of a saccharification rate and production efficiency, 1-240 hours are preferable, for example, Furthermore, 2-200 hours, and especially 3-140 hours are preferable.

  Thus, examples of the monosaccharide obtained by the hydrolysis reaction include glucose, fructose, mannose, galactose, xylose, and arabinose. Examples of the disaccharide include cellobiose, maltose, di-N-acetylchitobiose and the like, and oligosaccharides include those having 3 to 10 monosaccharide units. For example, cellotriose, cellotetraose, cello Pentaose, cellohexaose, maltotriose, maltotetraose, maltopentaose, maltohexaose, tri-N-acetylchitotriose, tetra-N-acetylchitotetraose, penta-N-acetylchitopentaose, Examples include hexa-N-acetylchitohexaose.

According to the method of the present invention, it is possible to produce a decomposing sugar from a polysaccharide at a saccharification rate of 60 to 100%, further 70 to 100%, particularly 80 to 100%. The saccharification rate was obtained by dividing the total mass of water-soluble sugars (monosaccharides and disaccharides typified by glucose) produced by the hydrolysis reaction by the mass of polysaccharides contained in the raw material subjected to the hydrolysis reaction. Say the value.
In addition, the yield (yield) of the decomposed sugar throughout all the steps is preferably 60% or more, more preferably 65% or more, and particularly preferably 70% or more from the viewpoint of production efficiency.
As described above, polysaccharides include many examples including cellulose, and cellulose includes α-cellulose having high crystallinity, modified β-cellulose and γ-classified as hemicellulose. There is also cellulose. In this application, the yield of polysaccharides, the saccharification rate, and the yield of decomposing sugars are based on α-cellulose, which is generally used as the cellulose purity in biomass. The method for measuring the amount of α-cellulose is described in the Examples.

  The obtained decomposed sugar can produce useful substances such as ethanol, polylactic acid, amino acids, xylitol, and erythritol by subjecting these decomposed sugars as a sugar source to microbial fermentation or chemical conversion.

<Measurement of α-cellulose amount>
Distilled water (150 mL), sodium chlorite (1.0 g) and acetic acid (0.2 mL) are added to about 2.5 g of the degreased product obtained by Sosley extraction, and heated on a hot water bath at 80 ° C. Addition of sodium chlorite and acetic acid and heating for 1 hour after the addition were further performed twice. The contents were filtered, and the filtration residue was washed with water and acetone and then dried. 1 g of the obtained dried product was added to 25 mL of 17.5% aqueous sodium hydroxide solution and left for 30 minutes. After 30 minutes, 25 mL of water was added, stirred for 1 minute, and allowed to stand for 5 minutes. The contents were filtered, and the filter residue was washed with 10% aqueous acetic acid and boiling water and then dried. The weight of the obtained dry residue was defined as the amount of α-cellulose.

<Measurement of water-soluble sugar concentration (glucose concentration, cellobiose concentration)>
Using a high performance liquid chromatograph manufactured by Hitachi, Ltd., a column Asahipak NH2P-50 4E (4.5 mmφ × 250 m) manufactured by Showa Denko was installed, and a gradient method was performed at a column temperature of 20 ° C. The mobile phase A solution was acetonitrile, and the B solution was 30% methanol water, and the solution was sent at 1.00 mL / min. The gradient conditions are as follows.
Time (min) A liquid (%) B liquid (%)
0 20 80
45 50 50
45.1 20 80
55 20 80
The sample injection amount was 5 μL, and detection was performed using a corona CAD detector manufactured by ESA Biosciences.

<Measurement of volume-based average particle size>
It measured with the laser diffraction type particle size distribution analyzer (the Beckman Coulter company make, LS13320).

<Calculation of α-cellulose yield>
The α-cellulose yield was calculated by the following formula (i).
α-cellulose yield (%) = {mass of α-cellulose contained in solid part obtained by solid-liquid separation of reaction product after heat treatment obtained in step (2)} / {in step (2) Mass of α-cellulose contained in pulverized raw material polysaccharide} × 100 (i)

<Calculation of saccharification rate>
The saccharification rate was calculated by the following formula (ii).
Saccharification rate (%) = {(water-soluble saccharide mass after reaction) − (water-soluble saccharide mass before reaction)} / mass of α-cellulose × 100 (ii)

<Calculation of decomposition sugar yield>
The degradation sugar yield (%) was calculated by multiplying the α-cellulose yield and the saccharification rate.

Example 1
100 g of dried rice straw was pre-ground with a grinder so that the volume-based average particle diameter was 3 to 5 cm. A vibrating rod mill (MB-1 manufactured by Chuo Kako Co., Ltd., 3.5 L vessel total volume) was filled with 13 rods with an outer diameter of 30 mm (filling rate 74%), and 100 g of pre-ground rice straw was charged, 1,200c By shaking for 10 minutes at / min, ground rice straw was obtained. The volume standard average particle size was 0.04 mm.
A slurry obtained by mixing 10 g of this ground rice straw (α-cellulose content 32% by mass) and 90 g of water was placed in a batch hydrothermal apparatus (Start 200 New Quick, volume 180 mL) manufactured by Nitto Koatsu, and the upper space was replaced with nitrogen and heated. did. The temperature rising rate was 3.4 ° C./min, and after reaching 200 ° C., it was immediately cooled. The time spent at 185 ° C. to 230 ° C. was 30 minutes. After cooling to room temperature, a solid part (solid residue) was obtained by filtration. The residue was fractionated so that the dry mass was 5% by mass, and 0.1 M citrate buffer solution having a pH of 5.0 was added to make a total of 10 g. 62.5 μL of cellulase (Novozymes Cell Crust 1.5 L) was added thereto for saccharification (addition of residue per 1 g of dry weight was 8.75 FPU). The saccharification reaction was carried out in a thermostatic bath at 50 ° C. for 48 hours while shaking at 110 r / min. After completion of the reaction, the liquid part was filtered with a 0.2 μm filter, and the water-soluble sugar concentration was measured. Cellobiose and glucose were measured as water-soluble sugars.

Example 2
A water-soluble sugar was obtained in the same manner as in Example 1 except that after reaching 200 ° C. in hydrothermal treatment, the mixture was cooled at 200 ° C. for 30 minutes and then cooled. The time spent at 185 ° C. to 230 ° C. was 65 minutes.

Example 3
A water-soluble sugar was obtained in the same manner as in Example 1 except that the batch hydrothermal apparatus was previously pressurized to 1 MPa with nitrogen gas.

Comparative Example 1
In the hydrothermal treatment, a water-soluble sugar was obtained in the same manner as in Example 1 except that it was immediately cooled after reaching 180 ° C.

Comparative Example 2
The ground rice straw obtained in Example 1 was enzymatically saccharified without hydrothermal treatment.

Comparative Example 3
A crushed rice straw was obtained by charging 40 g of dried rice straw into a grinder manufactured by Iwatani Corporation (Lab Miller 800DG, volume 260 mL) and stirring at 20,000 r / min for 10 minutes. The volume standard average particle diameter was 0.27 mm. 10 g of this ground rice straw (α-cellulose content 32% by mass) and 90 g of water were mixed, and then heat treatment and enzymatic saccharification treatment were carried out in the same manner as in Example 2 to obtain a water-soluble sugar.
Comparative Example 4
A ground rice straw was obtained in the same manner as in Comparative Example 3 except that stirring was performed at 20,000 r / min for 120 minutes. The volume standard average particle diameter was 0.05 mm. 10 g of this ground rice straw (α-cellulose content 32% by mass) and 90 g of water were mixed, and then heat treatment and enzymatic saccharification treatment were carried out in the same manner as in Example 2 to obtain a water-soluble sugar.
Table 1 shows the conditions and results of each Example and Comparative Example.

  As is clear from Table 1, in Examples 1 to 3, in which cellulose was pulverized with a vibration rod mill and then heat-treated at 185 to 230 ° C., the saccharification rate was high, and the yield of decomposing sugar throughout the entire process was high. It was. On the other hand, in Comparative Example 1 that was heat-treated at less than 180 ° C. and Comparative Examples 3 and 4 that were pulverized using a grinder instead of a vibrating rod mill, the α-cellulose yield after the heat treatment was high, but after the saccharification reaction As a result, the saccharification rate was low and the yield of decomposing sugar throughout the entire process was low. Moreover, the comparative example 2 which did not heat-process was a result that the saccharification rate is low and the yield of decomposition sugar is also low.

Claims (5)

Next steps (1) to (3):
(1) A step of applying a compressive shear stress to a polysaccharide to pulverize the polysaccharide to obtain a pulverized polysaccharide;
(2) A step of heat-treating the pulverized polysaccharide at 185 to 230 ° C. in the presence of water,
(3) A step of obtaining a solid part by solid-liquid separation from the reaction product after the heat treatment, and allowing a hydrolase to act on the solid part,
A process for producing monosaccharides, disaccharides and / or oligosaccharides.   The method for producing monosaccharides, disaccharides and / or oligosaccharides according to claim 1, wherein the time for heat treatment at 185 to 230 ° C is 10 minutes to 90 minutes.   The method for producing monosaccharides, disaccharides, and / or oligosaccharides according to claim 1 or 2, wherein compressive shear stress is applied to the polysaccharides using a vibration rod mill.   The method for producing a monosaccharide, disaccharide, and / or oligosaccharide according to any one of claims 1 to 3, wherein the heat treatment is performed at a pressure equal to or higher than a saturated vapor pressure of water.   The method for producing monosaccharides, disaccharides, and / or oligosaccharides according to any one of claims 1 to 4, wherein cellulose-containing biomass is used as the polysaccharide.