JP2009183264A - Saccharification method of rice straw - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a saccharification method of remarkably high efficiency, without performing chemical pretreatment using strong acid-strong alkali, having no problem of cost and environmental load which problem is caused by use of chemical agents, and only by easy operation, when using plant above-ground parts of rice plant. <P>SOLUTION: The saccharification method comprises recovering plant above-ground parts of rice plant containing starch of ≥5% based on dry weight in the stem and leaf parts, crushing, and performing saccharification treatment using an enzyme solution containing amyloglucosidase and β-glucan catabolic enzyme. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a saccharification method using an enzyme made from the above-ground part of rice plants.
Specifically, by using as a raw material the above-ground part of a rice plant containing 5% or more starch per dry weight in the foliage, and saccharifying using an enzyme solution containing amyloglucosidase and β-glucan degrading enzyme, The present invention relates to a saccharification method in which the efficiency of saccharification is significantly increased.

In response to the growing global needs for biofuels, competition for the development of bioethanol production technology derived from carbohydrate-based biomass is taking place on a global scale. In particular, it is considered that the development of utilization technology of lignocellulosic biomass that does not compete with food resources can be the most important breakthrough not only in Europe and the United States but also in Japan.
The saccharification technology development of lignocellulosic biomass has a history of about 200 years, but is now active again. In particular, enzyme saccharification technology centered on cellulase is attracting high expectations instead of saccharification technology centered on acid saccharification. However, in general, polysaccharides in lignocellulosic biomass are embedded in a cell wall having a complex structure, and development of a pretreatment step prior to the saccharification step has been considered essential.

Herbaceous biomass materials, mainly agricultural waste, are expected to increase the enzymatic saccharification efficiency by a mild pretreatment process compared to woody materials. To date, dilute sulfuric acid / steam explosion method The development of pretreatment technologies such as ammonia treatment has been vigorously conducted.
However, in these pretreatment steps, the costs associated with the use of chemicals and the problem of environmental impact have been pointed out, and the development of a milder and lower cost pretreatment technique is desired. In particular, with regard to the development of efficient saccharification technology using grasses and leaves of gramineous plants centering on rice straw, we are deeply involved in both agricultural policy and biomass energy policy in Japan, the United States, China, South Korea, Vietnam, Thailand, etc. We are attracting high expectations for being involved. In Japan, the development of bioethanol conversion technology for rice straw, which is the most abundant agricultural waste resource, is an urgent issue.

As a pretreatment technique for monocotyledonous plant cell walls such as rice straw, a relatively mild treatment method has been studied. For example, in the US Department of Agriculture project “Agricultural Initiative for Future Agricultural and Food Industry Systems (IFAFS)” conducted until 2005, a uniform sample of corn stover with component analysis data was supplied. We compared the usefulness of ethanol conversion technologies based on hand-held elemental technologies (pretreatment technologies such as dilute acid, water vapor, calcium hydroxide, ammonia, and ammonia explosion) and evaluated the characteristics of each technology (for example, Non-Patent Document 1). reference). Research has also been conducted on steam explosion methods for herbaceous materials.
However, all of these methods are pretreatments centered on either a reaction at a high temperature exceeding 140 ° C. or a reaction using a high concentration acid / alkali, and the cost associated with the advancement of the reaction tank, Problems such as acid / alkali recovery or treatment costs and environmental impact have not been solved.
Biomass saccharification is preferably performed in a mild manner so that it can be easily transferred to the fermentation process when fermentation of the saccharified product is as small as possible with little change in heat and pH. Corn starch saccharification technology that has already been put to practical use is composed of raw material grinding, heating at a relatively low temperature of around 100 ° C., and enzymatic saccharification steps (see, for example, Non-Patent Document 2). This technology is advantageous in that the investment in the reaction apparatus is suppressed and the use of chemicals is suppressed by a mild enzyme reaction, which is rapidly spreading for small and medium-sized bioethanol plants.
Currently, there is a demand for the development of such an easy saccharification technique using herbaceous biomass such as rice plants including rice.

Wyman, C. E., et al., Bioresour. Technol. 2005, 96: 1959-1966 Tomiaki Yamada, “Bio-Liquid Fuel” NTS Corporation (2007), pp.91-114

  The present invention solves the above-mentioned conventional problems, and when using the above-ground parts of rice plants as a raw material, the cost and environment associated with the use of chemicals without performing chemical pretreatment using strong acids and strong alkalis. It is an object of the present invention to provide a saccharification method that has no problem of load and has a remarkably high efficiency only by an easy operation.

  In the method of saccharification using the above-ground part of rice plant as a raw material, the present inventor uses the above-ground part of rice plant containing 5% or more of starch per dry weight of the foliage as a raw material, and amyloglucosidase and It has been found that the efficiency of saccharification can be remarkably increased by saccharification treatment using an enzyme solution containing β-glucan degrading enzyme, and the present invention has been completed.

The present invention relates to the following.
That is, the present invention according to claim 1 contains amyloglucosidase and β-glucan-degrading enzyme after recovering and pulverizing the above-ground parts of rice plants containing 5% or more starch per dry weight in the foliage. The present invention relates to a saccharification method characterized by saccharification treatment using an enzyme solution.
Moreover, this invention of Claim 2 is related with the saccharification method of Claim 1 which is a foliage part except the fruit which the said plant body above-ground part matured.
Moreover, this invention of Claim 3 is related with the saccharification method in any one of Claim 1 or 2 whose said plant body above-ground part is a thing of a heading stage to a mature stage.
Moreover, this invention of Claim 4 is related with the saccharification method in any one of Claims 1-3 in which the said plant body above-ground part contains a ground part.
Moreover, this invention of Claim 5 performs the heat processing at 40-130 degreeC, and / or performing a drying process within 24 hours, after collect | recovering the said plant body upper parts, The plant body above-mentioned part is a plant body cell of the above-mentioned plant body part and the sucrose, glucose and fructose utilization activity of microorganisms that inhabit the plant body and the surface thereof are suppressed. It relates to the saccharification method described in any one.
Further, the present invention according to claim 6 is the method according to claim 5, wherein the plant ground part in which the assimilation activity is suppressed is based on a dry weight of the plant ground part. The saccharification method according to claim 5, comprising 4% or more of sucrose, 0.5% or more of glucose, and 0.5% or more of fructose.
Moreover, this invention of Claim 7 is related with the saccharification method in any one of Claims 1-6 which collect | recovers 50% or more as glucose among the total amount of glucan contained in the said plant body above-ground part. Is.
Further, in the present invention according to claim 8, the β-glucan degrading enzyme is composed of amorphous cellulose, crystalline cellulose, and β- (1 → 3), β- (1 → 4) -glucan. It is related with the saccharification method in any one of Claims 1-7 which has the activity which hydrolyzes at least one of them.
In addition, the present invention according to claim 9 is characterized in that the enzyme solution containing the amyloglucosidase and β-glucan-degrading enzyme has both cellobiohydrolase and endoglucanase activities. It relates to the saccharification method described in any one.
The present invention according to claim 10 is characterized in that the enzyme solution containing amyloglucosidase and β-glucan degrading enzyme contains at least one of hemicellulose degrading enzyme, β-glucosidase, and α-amylase. It is related with the saccharification method in any one of claim | item 1 -9.
The invention according to claim 11 relates to the saccharification method according to any one of claims 1 to 10, wherein the amyloglucosidase and / or β-glucan-degrading enzyme is a microorganism-derived enzyme or enzyme preparation. It is.
Moreover, this invention of Claim 12 performs the heat processing of 70-130 degreeC before the said saccharification process and / or in the said saccharification process, The saccharification in any one of Claims 1-11 It is about the law.
Moreover, this invention of Claim 13 is related with the manufacturing method of ethanol characterized by performing ethanol fermentation using the saccharified liquid obtained by the saccharification method in any one of Claims 1-12. It is.

The present invention is a simple operation that does not involve chemical pretreatment using a strong acid / strong alkali when using the above-ground plant part of rice as a raw material, and does not have a problem of cost and environmental burden associated with the use of chemicals. It is possible to perform saccharification with remarkably high efficiency, particularly to produce glucose. Specifically, the present invention makes it possible to saccharify 50% or more of the total amount of glucan contained in the raw material.
In addition, the present invention leads to the development of efficient biomass saccharification technology with low cost and low environmental impact, and in particular, development of bioethanol production technology, which is an urgent issue not only in Japan but around the world. It is possible to provide biomass saccharification technology that is extremely important as a new innovation.

  The present invention relates to a method for saccharification using an above-ground part of rice plant as a raw material, wherein the above-ground part of rice plant containing 5% or more of starch per dry weight in the stem and leaf is used as a raw material, and amyloglucosidase and β -It relates to a saccharification method in which the saccharification efficiency is remarkably enhanced by saccharification treatment using an enzyme solution containing a glucan-degrading enzyme.

  “Saccharification” refers to hydrolysis by cleaving a glycosidic bond of a polysaccharide or oligosaccharide to lower the molecular weight to a oligosaccharide or monosaccharide having a lower polymerization degree. In the present invention, it means to decompose to glucose, maltose, isomaltose, maltooligosaccharide, isomaltoligosaccharide, cellobiose, cellooligosaccharide, and preferably to glucose.

In the present invention, the starch content can be measured as a monosaccharide by subjecting starch to heat gelatinization and then subjected to α-amylase and amyloglucosidase treatment, and can be analyzed using, for example, Total Starch Kit of Megazyme.
In addition, it is unclear whether the starch contained in the shoots and leaves of rice mainly has an amylose / amylopectin structure peculiar to stored starch, and there is a possibility that it has a glycogen-like structure.
In the present invention, for convenience, a polymer that can be saccharified by α-amylase and amyloglucosidase is defined as “starch”.

  In the present invention, the total amount of glucan is calculated from the amount of glucose (glucose amount ÷ assuming that the total glucose obtained as a hydrolyzate of the above-ground plant body of rice as a raw material is a linear polysaccharide of glucose as a basic skeleton. 180 × 162).

Further, one of the objects of the present invention is to utilize biomass raw materials that are not used for food in order to avoid competition with food. It is desirable to be "excluding mature fruit (rice)".
Of rice, those that are not intended for food use, that is, those that are intended for feed rice or energy rice, and the reproductive organs such as seeds and flowers in the ear do not correspond to food. If so, “the above-ground part of the plant body containing mature fruits” can be used as a raw material for the saccharification method of the present invention.
In addition, rice that is not mature, such as rice at the heading stage, can efficiently transport, store, and pulverize raw materials by using the whole plant part without separating the ears and seeds. It becomes possible.

As rice varieties that can be used in the present invention, Oryza sativa and those based on it can be widely used.
Preferably, the kind which contains many starches in a foliage part is preferable. The starch content depends not only on the cultivar but also on the cultivation method, and examples include varieties such as japonica type fukuhibiki, Hokuriku 187, Hokuriku 184, Dontokoi, Indica type Habataki, Takanari, etc. be able to.

The above-ground part of the plant body of rice that can be used as a raw material in the present invention is only required to contain 5% or more, preferably 20% or more of starch in the “stems and leaves” per dry weight.
In addition, the case where a mixture containing starch exceeding 5% per dry weight and a mixture containing less than 5% starch per dry weight is used in the present invention when the average value of the whole is 5% or more. Included as raw material.
If the technique of the present invention is used, about 170 g or more of glucose is expected to be recovered from 1 kg of the above-ground part of rice plants containing 5% or more starch in the foliage. In addition, it is possible to improve the glucose recovery efficiency by optimizing the enzyme reaction conditions by a known method.

The “plant plant above-ground part” that can be used in the present invention refers to tissues such as stems and leaves, mature fruits (rice), and ears of inflorescences of rice.
In addition, the “stem and leaf part” in the present invention refers to a tissue or a leaf centered on a leaf sheath part or a heel part, and includes a part of the underground part mixed when it is pulled out.

It is known that the amount of starch contained in the rice foliage varies depending on the growth stage. For example, the following is specifically described for rice.
A small amount of starch is contained in the stems and leaves excluding the rice ears after rice harvest, but when normal mowing and air drying at room temperature, the starch content is around 0-2% of the dry weight Sometimes.
The dry weight is calculated from the weight of the residue when water is evaporated. For example, it can be determined as the weight when dried at about 105 ° C. and almost constant weight.

  In addition, the raw material used for this invention may be described as "rice straw". The so-called “rice straw” is sometimes referred to vaguely as “the plant body above the matured fruit (rice) of the harvested rice”. In the present invention, “rice straw” refers to the plant of rice. The above-ground part is harvested and collected, and refers to the whole part of the plant body including the heads and seeds in the heading stage and the mature stage, and the above-ground part of the plant body excluding the mature fruit harvested at the time of rice harvest. Is. These are not only those that have just been cut, but also those that have been cut and then dried.

The starch contained in the stem and leaves of rice exceeds 20% at the heading stage and is highly important as a carbohydrate (He, Y., H., et al., Plant Prod. Sci. 2005, 8: 546-552.).
This phenomenon has been studied for a long time from the viewpoint of the relationship between starch accumulation in fruits and the amount of starch accumulated in the foliage and its time (for example, Nagata, K., et al., Plant Prod. Sci. , 2001, 4, 173-183. Most of this starch shifts to the ear as it matures, but there are some rice varieties that leave about 5% to 20% or more of starch in the foliage even at the maturity stage. Some are accumulated. (For example, Hiromichi Yamaguchi & Osamu Matsumura, Nisakuki, 2004, 73, 402-409.).
Specific examples include varieties such as Japonica type Hokubiki, Hokuriku 187, Hokuriku 184, Dontokoi and the like, which are particularly suitable for use in the present invention.
The accumulation of starch in the foliage of rice depends not only on the variety but also on the cultivation method. Therefore, even when using various rice varieties other than the above, fertilization management etc. By using the cultivation technique described above, it is possible to cultivate rice having a stem and leaf portion of rice that has accumulated an amount of starch that can be used as a raw material of the present invention.

Normally, it is known that the starch accumulation amount in the stem and leaf part of rice reaches a peak near the heading stage. Therefore, it is desirable to collect rice stems and leaves from before and after heading to maturity.
Grains are obtained at maturity, but starch derived from the stem is reduced by translocation to the ear. However, there are cases where the amount of starch increases again in the mature period. Ordinary grain starch is a starch grain of about several μm composed of amylopectin tufted particles and amylose, and the enzymatic degradation of raw starch is low.
On the other hand, the starch in the stem and leaves at the heading stage has a highly enzymatic degradable structure that assumes sugar translocation to the head after the heading stage, which leads to the starch saccharification technology using amyloglucosidase in the present invention. It is thought that.
In addition, there are many amorphous β-glucans such as β- (1 → 3) and β- (1 → 4) -glucan when secondary wall differentiation is not progressing, mainly around the heading period. As is known, β- (1 → 3) and β- (1 → 4) -glucan in the heading stage are present in a state where they are susceptible to degradation by their degrading enzymes, and this is effective in the present invention. It is thought to contribute to glucose production.

  On the other hand, in the mature stage where the starch content is high, the decomposition rate of the non-starch glucan tends to be maintained high. That is, there is a merit in performing enzymatic saccharification of the non-starch glucan part contained in the above-ground part of the plant body of the stem and leaf part having a high starch content. The cause of this is unknown, but the survival rate of the conductive tissue is relatively high in the foliage at the stage where much starch remains in the stem part around the ground part and the translocation function remains, and the tree part There is a possibility that the progress of crystallization will remain to some extent, or there may be a non-starch glucan that correlates with the amount of starch.

  When using rice straw harvested by harvesting and harvesting mature rice with a high starch content in the foliage as a raw material, saccharification can be easily achieved with 50% or more of the glucan content. Specifically, about 160 mg or more of glucose can be recovered from 1 g of rice straw.

In addition, the “border part (ground part from 0 to about 15 cm from the ground)”, which is a part of the rice near the ground, contains a large amount of starch. However, rice straw collected by a harvesting operation using a normal mower (a normal mowing method) has a low ratio of the portion of the ground, and the average starch amount is low.
Therefore, rice straw having a high starch content in the stalks and leaves per dry weight can be obtained by harvesting with a recovery method including the ground part.

In general, it is known that sucrose, glucose, and fructose are present as free carbohydrates in plants.
For example, He, Y., H., et al., Plant Prod. Sci. 2005, 8: 546-552, show metabolism related to the production and degradation of sucrose, glucose and fructose in the leaf sheath.
In addition, analytical data indicate that about 35% of cellulose is present in rice straw (for example, Saha, BC, J. Ind. Microbiol. Biotechnol., 2003, 30, 279-291). However, attention has not been paid to the existence and utilization of “free sugars” contained in rice straw, and biomass conversion research has not been conducted.
In general, rice straw is a temperature at which assimilation activity of the cells of the plant body and microorganisms that inhabit the plant body and the surface is maintained while it is gradually dried by sun drying or field stacking after harvesting. Often left for a long time under conditions of humidity and humidity. Therefore, it is considered that easily degradable free carbohydrates are reduced in rice straw obtained by a general method.

In the saccharification method of the present invention, it is desirable that the above-ground part of the rice plant as the raw material contains sucrose, glucose and fructose as “free carbohydrates”.
Among these free carbohydrates, the amount of fructose contained in the above-ground parts of rice plants is determined by an analysis method using the Roe method (Sakuzo Fukui, Biochemical Experiment Method 1 "Quantification Method for Reducing Sugar", ISBN4 -7622-0102-2) can be applied mutatis mutandis to determine the total amount of fructose in water extract from above-ground parts of rice plants.
Here, the “total amount of fructose” in the water extract of the above-ground part of rice plants is a value reflecting the amount of free fructose and the amount of fructose residues in the sucrose molecule.
Further, the “free glucose amount” can be measured by a glucose determination method by Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.).
Note that the “amount of sucrose” can be estimated by using the measured values of the total fructose amount and the free glucose amount. That is, since the amount of free fructose in the plant body is approximately the same value as the amount of free glucose, the amount of sucrose can be estimated by estimating the amount of fructose in the sucrose molecule.
Specifically, the amount of each free carbohydrate can be estimated by applying the following equation.
First, the amount of free fructose can be estimated by the following formula (I).

  The amount of sucrose can be estimated by the following formula (II).

  And the total amount of free carbohydrates can be estimated from the value of the amount of each free carbohydrate measured and estimated as described above by the following equation (III).

  That is, the total amount of free carbohydrates in the present invention can be determined as the sum of the amount of sucrose, the amount of free glucose and the amount of free fructose.

In the present invention, it is desirable that the above-ground part of the rice plant as a raw material is subjected to “treatment for suppressing the assimilation activity of the cells of the plant body and the microorganisms inhabiting the surface of the plant body” as soon as possible after harvesting. .
The treatment for suppressing the assimilation activity can be performed by performing at least one of heat treatment and drying treatment. Preferably, performing the heat treatment, more preferably performing the combination of both, can improve the effect of suppressing the assimilation activity, and is suitable. Moreover, after performing a drying process, it is desirable to preserve | save so that the dry state of the said raw material may be maintained.

The treatment for suppressing the assimilation activity should be carried out as soon as possible after the above-ground part of the plant of rice as a raw material is recovered, specifically within 24 hours, preferably immediately after harvesting or threshing.
The rate at which the free carbohydrates in the raw material are assimilated varies depending on the temperature and moisture content of the recovered raw material, but by performing these treatments within 24 hours, the conversion characteristics of the raw material (biomass raw material) Is expected to remain high.

In addition, when heat treatment is performed in the treatment for suppressing the assimilation activity, the heating is performed at a relatively low temperature of 40 to 130 ° C., desirably 50 to 75 ° C., more desirably 50 ° C. or more and about 70 ° C. Heat.
In addition, when performing the saccharification method of this invention by a commercial process, it can carry out by the steam spraying which can heat-process a lot of raw materials efficiently. A typical steam spraying temperature used in a commercial process is about 130 ° C., which is the upper limit temperature.
The treatment time of the heat treatment largely depends on the treatment temperature, but at a temperature of 100 ° C. or less for a few seconds to 120 hours, for about 1 minute to 72 hours, the free carbohydrates in the raw material are contributed. Most of the cells of the plant body and the microorganisms that are transformed can be killed. In addition, it is possible to use a repeated heat treatment method in consideration of spore germination.

In addition, when it is set as the process which suppresses the said assimilation activity by heat processing which does not fully dry, after treatment, microorganisms invading from the outside or a dormant microorganism grows in the form of spores, etc. In the meantime, there is a risk of the utilization of free carbohydrates. Therefore, when storing, it is desirable to store the raw material isolated from the outside after sterilizing spores present in the raw material, or to perform a drying treatment after the heat treatment.
The raw material after the heat treatment (biomass raw material) should be transferred to the saccharification process immediately after storage for a period that prevents the decomposition of free carbohydrates by microorganisms, while preventing the entry and generation of microorganisms from the outside. It is. In particular, when heat treatment is performed under conditions that exceed the gelatinization temperature of starch, starch absorbs water and the degradability is improved, so that not only free sugars but also utilization of starch is promoted after invasion of microorganisms. It becomes.

In addition, in the treatment for suppressing the assimilation activity, when a drying treatment is performed, a microorganism that assimilate free carbohydrates in the raw material by lowering the water content to 20% or less, preferably about 5% or less. Growth is greatly suppressed.
In addition, although the said drying process may be performed by air-drying a raw material indoors or in the sun, the said raw material can be dried in a short time by performing hot-air drying or heat drying in a thermostat. be able to. At the same time, the heat treatment can be performed simultaneously.
As described above, after the drying process is performed, it is desirable to store the raw material so that the dried state is maintained.

In this step, by performing the treatment for suppressing the assimilation activity as described above, the assimilation activity of the cells and microorganisms of the plant body can be sufficiently reduced, and glucose, which is a free carbohydrate contained in the raw material, Utilization of fructose and sucrose can be suppressed.
In addition, by this treatment, the above-ground part of the plant body of rice as the raw material contains these free carbohydrates, and is 5% or more, preferably 6% or more of the dry weight, specifically, in the mature stage. The free carbohydrate can be accumulated in an amount of about 7% or more of the dry weight.

  Thus, by passing through this step, the plant body above-ground part of the raw material rice can be made into a raw material containing a large amount of free carbohydrates, so that the saccharification efficiency of the saccharification method of the present invention is greatly improved. It is possible to improve the quality of rice straw as a biomass raw material.

In the method of saccharification by using the above-ground part of the plant body including the stem and leaf part of rice as a biomass raw material, research using the above-ground part of the plant body (so-called “rice straw”) excluding the mature fruit of the harvested rice is conducted. I have been.
Conventionally, when saccharification is performed using rice straw as a raw material, pretreatment using high heat, pressure, acid, alkali, or the like has been required.
Its purpose is to remove and dissociate lignin, remove esters attached to hemicellulose, or modify the tissue structure suitable for enzymatic saccharification, leaving as much saccharification substrate cellulose and hemicellulose as possible. The purpose is to improve the efficiency of the next stage of enzymatic saccharification.
However, rigorous pretreatment using high heat, pressure, acid, alkali, etc., elution and modification of saccharification substrate, acceleration of lignin deposition and side reactions, concomitant wear of useful substances, fermentation inhibition and harmful substances The generation of is induced.
In addition, there has been a concern that problems in processing costs and work safety management may occur in the manufacture of heat-resistant / pressure-resistant containers, heating / pressurizing processes, acid / alkali processing processes, recovery / disposal processes, and the like.

In addition, rice that contains a large amount of starch in the foliage has attracted attention from the viewpoint of its use as feed rice, and as a quality evaluation technique for predicting “feed digestibility”, a quantitative technique after enzymatic degradation has been developed. ing. For example, the report by Hiroshi Matsuyama et al. (Livestock Pasture Research Result Information, 2004 No. 3, pp. 51-52) can be mentioned as a result.
However, from the viewpoint of directly “saccharifying” biomass (producing monosaccharides such as glucose and low-polymerization sugars), the above-ground part of rice plants that have accumulated starch at high concentrations in the foliage (rice straw) There are no reports on the hydrolysis characteristics of these enzymes.

  The present invention is a method of saccharification using the above-ground part of rice plant as a raw material, without performing chemical pretreatment using strong acid / strong alkali, and there is no problem of cost and environmental burden associated with the use of chemicals, The present invention relates to a saccharification method using an enzyme that has a remarkably high saccharification efficiency.

  In the present invention, the saccharification treatment using an enzyme refers to saccharification of the above-ground part of the rice plant, which is the raw material, using an enzyme solution containing amyloglucosidase and β-glucan degrading enzyme.

When the enzyme is directly acted on the above-ground part of the rice plant, which is the raw material, immediately after it is cut off from the roots, it is moderately pulverized (including cutting) because the enzyme penetration efficiency is low. Grinding increases the surface area and promotes subsequent enzyme reactions.
The crushing can be performed by a method such as cutting with a rotary blade, crushing with a roller, grinding with a stone file, or crushing with a hammer mill.

In the present invention, the raw material can be dissolved using a cell wall lytic enzyme to increase the surface area. Since pulverization inevitably improves the reaction efficiency, there are no factors that limit the pulverization conditions as long as cost, energy efficiency, work efficiency, and environmental load are taken into consideration. It is desirable that a size of several tens of μm or more is a main component. If the particle size is small, the surface area increases, but adverse effects such as an increase in the pulverization cost, a decrease in workability due to dust, and thermal deterioration occur.
Depending on the drying state of the raw material, drying is performed at a temperature of 70 ° C. or less before or after pulverization as necessary, whereby the pulverization efficiency by the machine is improved and the data is reproducible.

In addition, when the wet substrate (raw material powder) or the suspension of the substrate (raw material powder) is heated at a temperature of 70 ° C. or higher, preferably 80 ° C. or higher, the starch becomes a water-absorbing gelatin and enzymatic saccharification of rice straw Increases speed. In addition, when performing the saccharification method of this invention by a commercial process, it can carry out by the steam spraying which can heat-process a lot of raw materials efficiently. A typical steam spraying temperature used in a commercial process is about 130 ° C., which is the upper limit temperature. Therefore, the heat treatment temperature is a temperature in the range of 70 ° C to 130 ° C, preferably 80 ° C to 130 ° C.
The heat treatment time is affected by the heat exchange rate and the amount of water, but for a few seconds to several tens of minutes, specifically for 5-30 minutes until the starch is fully gelatinized. It is desirable to do.
In the present invention, it has been found that when only the final starch conversion amount is considered, this heat treatment step is not necessarily required. However, there is no reason to hinder the introduction of the heat treatment step in that the activity of the enzyme is increased and the reaction time is shortened.

In the heat treatment, in order to gelatinize the starch, when the substrate (raw material powder) is heated in a wet state, water is used for wetting or suspending the substrate (raw material powder). It is possible to use water to which an acid or a dilute alkali having a pH of 12 or less is added.
By performing the heat treatment under such conditions, it is possible to sufficiently reduce the cost for adjusting the pH to 3 to 7 when performing subsequent enzymatic saccharification, and to promote gelatinization of starch In addition to this, a slight enzyme saccharification promoting effect is expected.
By performing the heat treatment at the predetermined temperature in the presence of a dilute acid, a slight improvement in the efficiency of the subsequent enzyme treatment due to partial decomposition of xylan or structural change of silica can be expected in addition to starch gelatinization. Further, by performing the heat treatment at the predetermined temperature in the presence of a dilute alkali, in addition to starch gelatinization, decomposition of acetyl ester of xylan or structural change or release of lignin, partial release of silica, etc. A slight improvement in enzyme treatment efficiency can be expected.

When the heat treatment is performed using water to which a dilute acid or dilute alkali is added, hydrochloric acid, sulfuric acid, or the like can be used as the acid, and sodium hydroxide, calcium hydroxide, water can be used as the alkali. Potassium oxide, ammonia, etc. can be used.
In addition, after the heat treatment using the diluted acid or the diluted alkali, the added acid or alkali is volatilized or the pH is adjusted to 3 to 7 by adding an alkali or an acid, respectively. desirable. This can avoid the loss of gelatinized starch and free carbohydrates.
In addition to the dilute acid and dilute alkali as described above, in which a chemical treatment effect is expected, a buffer solution or a weak acid solution adjusted to a pH of about 3 to 7 suitable for enzyme use is used in advance. Then, heat treatment for starch gelatinization can be performed.
In addition, if an oxidizing agent such as oxygen, ozone, or hydrogen peroxide is used during the heat treatment, lignin release is promoted, so that enzymatic saccharification efficiency can be improved.

In addition, it is desirable to use thermostable α-amylase during the heat treatment. The thermostable α-amylase is not easily inactivated even at a high heat treatment at around 100 ° C.
If this enzyme is used at the time of heat treatment, starch that has been gelatinized in the heat treatment is rapidly digested, and handling properties are improved by reducing the viscosity.
The saccharification treatment with amyloglucosidase, which is a feature of the present invention, does not prevent the introduction of the existing liquefaction technology with α-amylase and the use of a debranching enzyme with pullulanase or isoamylase.

  In addition, the heat treatment may be performed before the saccharification treatment or during the saccharification treatment. Preferably, when all of the saccharifying enzymes have heat resistance, heating is performed simultaneously with the saccharification step, and when at least a part of the saccharifying enzyme is deactivated during the heat treatment, heating is performed before the saccharification treatment. It is desirable to carry out enzymatic saccharification after cooling to a temperature at which the enzyme acts stably.

In the present invention, the enzyme solution containing amyloglucosidase and β-glucan-degrading enzyme contains amyloglucosidase and β-glucan-degrading enzyme.
In addition, it preferably contains at least one of hemicellulose-degrading enzyme, β-glucosidase, and α-amylase.
In the present invention, the enzyme solution containing amyloglucosidase and β-glucan degrading enzyme preferably has both cellobiohydrolase and endoglucanase activities.

The amyloglucosidase (= glucoamylase) in the present invention is an α-glucan degrading enzyme, which hydrolyzes α- (1 → 4) bond or α- (1 → 6) bond from the non-reducing terminal side of starch. It is an enzyme.
Specific examples include those derived from the genus Aspergillus and those derived from the genus Rhizopus.
In addition, the amyloglucosidase in the present invention can be increased in efficiency by increasing the number of non-reducing terminals when used in combination with an endo-hydrolyzing enzyme such as α-amylase, pullulanase or isoamylase.
Some enzyme preparations such as cellulase preparations exhibit α-amylase activity, and it is considered that glucose production (saccharification) by amyloglucosidase is promoted by the action of such enzymes.

In general, raw starch is known to be composed of a fraction having high enzyme digestibility and a fraction that is difficult to digest, and the latter varies depending on the origin of starch and the processing steps. Starch is water-absorbed and gelatinized by heating starch in water, improving the digestibility of indigestible fractions. However, when gelatinized in a matrix such as a tissue fragment, the gelatinized starch remains in the matrix and is recovered. There is a risk of falling.
Therefore, conventionally, for efficient saccharification of starch, a method of heating to around 95 ° C. in a state containing heat-resistant α-amylase using a jet cooker or the like as appropriate and liquefying while gelatinizing has been used. It was.
In addition, in the standard total starch analysis method (Megazyme Total Starch Kit = AOAC method 996.11, AACC method 76.13), a ground sample is dispersed with 80% ethanol, heat-resistant amylase is added and immediately heated at 100 ° C. Starch is liquefied. There is also a saccharification method using amyloglucosidase alone, but it involves a preliminary treatment with 80% ethanol and a heat treatment for several hours, which is a complicated process. (He, Y., H., et al., Plant Prod. Sci. 2005, 8: 546-552., Supra.)

  Under such circumstances, it has been found that gelatinization of starch is not always necessary in the present invention. Moreover, it discovered that it can apply also to the raw material of this invention that most is rapidly and efficiently converted into glucose by saccharification of starch using amyloglucosidase (= glucoamylase). In addition, conversion characteristic data for the purpose of saccharification of the above-ground part of rice plants (rice straw) containing a large amount of starch in the foliage has not existed so far. The decomposition characteristics of starch were not elucidated.

The saccharification treatment in the present invention is characterized in that in the saccharification treatment of the raw material, amyloglucosidase decomposition is performed and decomposition by β-glucan degrading enzyme is performed.
The β-glucan-degrading enzyme here indicates hydrolysis activity for β-glucan such as cellulose, β- (1 → 3), β- (1 → 4) -glucan, and xyloglucan in the raw material. Enzymes are widely shown, and among them, enzymes showing higher activity against substrates other than cellulose will be included.
In addition, enzymes that cleave β- (1 → 3), β- (1 → 4) -glucan and xyloglucan that are widely present mainly in seedlings of gramineous plants also correspond to β-glucan degrading enzymes.

As a conventional technique, a lichenase that hydrolyzes a β- (1 → 3) bond of β- (1 → 3), β- (1 → 4) -glucan is particularly effective when used in combination with β-glucosidase. Glucose can be produced. This technique is known as the AACC method (32-23) or the AOAC method (995-16), but is a technique mainly for barley, wort and the like.
However, β- (1 → 3) and β- (1 → 4) -glucan are known to decrease with the growth of the plant, but the above-ground part of rice plants containing a large amount of starch in the stem and leaves. Information on biomass saccharification, such as the balance of various polysaccharides in the total glucan, the abundance and degradation characteristics of β- (1 → 3), β- (1 → 4) -glucan, etc. The value as a biomass component has not been noticed.

Generally, the above-ground part of rice plants after harvesting rice has about 30% glucan in dry weight. Assuming a fermentation efficiency of 90% and a distillation efficiency of 100%, when producing bioethanol using only the glucose in rice straw as the fermentation raw material, the 9 kg, 8 kg, 7 kg, 6 kg, 5 kg or 4 kg levels are dried. In order to produce 1 liter of ethanol using rice straw, it is necessary to hydrolyze about 156 g, 174 g, 196 g, 224 g, 261 g or 314 g or more of glucan from 1 kg of rice straw to convert it into glucose. .
Therefore, in addition to studies to improve the yield of glucose derived from cellulose, a method for producing ethanol using xylose obtained by saccharifying xylan contained in dry rice straw as a fermentation raw material has been energetically used. It is being considered.
However, the method using recombinant microorganisms for xylose fermentation requires the development of a highly-managed fermentation / waste treatment facility compared to a fermentation facility using normal yeast. It is desired to perform fermentation using glucose as a substrate using non-recombinant yeast.

In the saccharification method of the present invention, not only starch contained in the raw material as a biomass raw material but also non-starch glucan contained in the raw material can be saccharified and recovered as glucose.
In particular, the saccharification method of the present invention can recover 50% or more of the total glucan contained in the above-ground part of the rice plant as glucose.

Moreover, in the saccharification method of this invention, the saccharification efficiency of the glucan contained in a raw material can be improved by using the above-ground part of the rice plant body which contains a large amount of starch in a stem and leaf part as a raw material.
The mechanism of the relationship between starch and glucan hydrolysis efficiency contained in the raw material is not clear, but as in the mature stage, the present inventor is obtained from a variety line at the heading stage rich in starch, We found that non-starchy glucan tends to be less in the above-ground part of rice after harvesting rice (so-called “rice straw”), and there is a non-starch easily degradable glucan fraction in the mature stage, It was found that the amount was almost constant regardless of starch content. These tendencies are the same as those in the mature period, and rice straw with a higher starch content has a higher rate of decomposition of non-starch glucan, indicating the presence of easily degradable glucan in non-starch glucan. This is considered to be due to the high ratio and the low abundance ratio of the hardly degradable glucan containing crystalline cellulose as a main constituent.
The present inventor has also found that the amount of β- (1 → 3) and β- (1 → 4) -glucan in heading season is higher than that in mature rice straw. There is a large amount of β- (1 → 3), β- (1 → 4) -glucan in the raw material, and the use of β-glucan degrading enzyme using this as a substrate may increase the amount of glucose produced. Can be considered.
Further, it was suggested that an enzyme in a cellulase preparation such as endoglucanase may increase the amount of glucose produced by using it as a substrate.
Heretofore, the interaction between β- (1 → 3), β- (1 → 4) -glucan contained in the raw material and other components and the susceptibility to direct enzymatic degradation have not been studied at all. .

In the present invention, β-glucan-degrading enzyme means hydrolyzing at least one of amorphous cellulose, crystalline cellulose, and β- (1 → 3), β- (1 → 4) -glucan. It has activity to do.
Specifically, cellobiase, which is “β-glucosidase”; cellulose, cellobiohydrolase, endoglucanase; “hemicellulose-degrading enzyme”, xylanase, β-D-xylosidase, α-L- Examples include arabinofuranosidase, acetyl xylan esterase, feruloyl esterase; lichenase, which is a degradation enzyme of β- (1 → 3), β- (1 → 4) -glucan.

  As the enzyme solution used for the saccharification treatment in the present invention, a β-glucan degrading enzyme containing at least β-glucosidase and cellulase can be used. In addition, it is preferable that those containing β-glucosidase, cellulase and hemicellulose-degrading enzyme, and those containing β-glucosidase, cellulase and lichenase are preferable.

Moreover, as said cellulase, it is desirable to contain what has cellobiohydrolase activity and what has endoglucanase activity.
That is, the enzyme solution in the present invention preferably has cellobiohydrolase activity or endoglucanase activity, and further has both cellobiohydrolase activity and endoglucanase activity. It is desirable.

Many commercially available cellulase preparations contain degrading enzymes for various β-glucans including cellulose. For example, “Celluclast 1.5L” from Novozymes Japan is β- (1 → 3), The hydrolysis activity of β- (1 → 4) -glucan and xyloglucan is strongly detected. In addition, the presence of a plurality of enzymes may enable saccharification more effectively than a single enzyme.
Here, what has activity as a cellobiohydrolase or an endoglucanase is demonstrated as the example. Cellobiohydrolase effectively degrades crystalline cellulose, and endoglucanase effectively degrades amorphous cellulose. In the present invention, the saccharification efficiency can be further increased by using both of them together.

The β-glucan degrading enzyme contained in the enzyme solution used for the saccharification treatment of the present invention contains at least β-glucosidase and cellulase.
Since cellulose may produce a large amount of cellobiose by cellulase, the use of β-glucosidase is effective for increasing the yield of glucose.
That is, β-glucosidase is effective for hydrolyzing cellobiose as a substrate to produce glucose. Further, it is effective for suppressing a decrease in the activity of cellulase due to the accumulation of cellobiose which is a reaction product of cellulase.
In addition, when using a saccharification product by the saccharification method by this invention as a raw material of bioethanol, it is effective also in the meaning which converts cellobiose which a yeast cannot assimilate to assimilating glucose.

In order to obtain a hemicellulose-derived monosaccharide such as xylose, it is also effective to add a hemicellulose-degrading enzyme system.
Examples of hemicellulose-degrading enzymes include xylanase, β-D-xylosidase, α-L-arabinofuranosidase, acetyl xylan esterase, and feruloyl esterase. Some of these can be used, or an enzyme preparation produced by a microorganism and containing one or more hemicellulose-degrading enzymes can be used. For example, “Viscozyme L” from Novozymes Japan is an enzyme preparation.

  Furthermore, in order to increase saccharification efficiency, by decomposing other components that constitute the cell wall such as lignin, a new substrate surface in the raw material is exposed, and saccharification by other enzymes used in other saccharification treatments is promoted. can do.

In addition, sucrose existing in the above-ground parts of rice plants is directly assimilated by many fermenting microorganisms such as yeast. However, if it is necessary to increase the yield of glucose, a shoe such as invertase can be used. It is effective to use Claus hydrolase.
In the conventional method of performing chemical pretreatment assuming the enzymatic saccharification of cellulose, cellulose is recovered as an insoluble product, and the liquid containing the chemical solution used for chemical pretreatment is removed. However, with the removal of the chemical solution, soluble sucrose is washed away, resulting in sugar loss.
On the other hand, in the saccharification method according to the present invention, the pulverized raw material is used to obtain a saccharified solution directly after appropriate heat treatment (because no operation for removing the liquid is required), and thus included in the pulverized raw material All the soluble sucrose can be recovered.

In the present invention, when the saccharification treatment is performed, the concentration of the pulverized material in the above-ground part of the rice plant as the raw material is about 1% (w / v) to about 30% (w / v) in dry matter calculation. Preferably, it is desirable to set it to about 5% (w / v) to about 20% (w / v).
When it is intended to obtain a sugar solution using a pulverized product of about 3 mm mesh pass or less, the enzyme reaction time in the saccharification treatment is about 30 minutes to 48 hours, preferably 30 minutes to 6 hours. Is desirable.
Moreover, the temperature of the enzyme reaction in a saccharification process is 20-60 degreeC, Preferably it is 30-50 degreeC. The pH of the enzyme reaction in the saccharification treatment is between pH 3 and 7, preferably between pH 3.5 and 6.

The essential part of the present invention is that high saccharification efficiency can be obtained by the action of amyloglucosidase and β-glucan degrading enzyme, which are α-glucan degrading enzymes.
The supply sources of these enzymes include various possibilities such as enzyme preparations, purified enzyme preparations, microbial culture solutions, and products of microorganisms outside, inside or on the surface of the cells. Can be used.
The present invention is also applicable to the case where a fermented microorganism capable of producing at least a part of these enzymes is cultured and subjected to fermentation simultaneously with saccharification. Preferably, the microorganism-derived enzyme or enzyme preparation is used as a reaction solution. It is preferable to add to and use.

In addition, as a raw material of the present invention, when the above-ground part of a rice plant from the heading stage to the mature stage is used as a raw material, the saccharification treatment liquid also contains a hemicellulose degradation product, and microorganisms such as pentose fermentation yeast It becomes possible to convert into useful substances, such as ethanol.
Furthermore, fermentation residues such as ethanol by the microorganism include lignin residues, hemicellulose and cellulose residues, and enzyme-derived proteins, which can be used as feed, fertilizer, fuel, or for further chemical or biological use. Chemical conversion allows extraction of chemicals or bioenergy.
In the conventional method of chemical pretreatment using strong acid / strong alkali, lignin is chemically modified by severe pretreatment with sulfuric acid etc. Although its commercial value is reduced, the saccharification method of the present invention can minimize alteration of the by-product.

According to the saccharification method of the present invention, it is possible to obtain a saccharified solution from a raw material in a gentle manner, and ethanol can be produced by performing ethanol fermentation using the obtained saccharified solution.
As described above, the saccharification method in the present invention does not use highly reactive chemicals such as strong acid and strong alkali, so ethanol fermentation such as furfural and 5-hydroxymethylfurfural as a by-product by sulfate saccharification is performed. Not only is the production of the inhibiting substance suppressed, but the pH can be adjusted easily. That is, using the saccharified solution obtained by the saccharification method of the present invention for ethanol fermentation has great advantages in terms of ease of operation, efficiency and cost.
Moreover, in the saccharification method of this invention, since heat processing are performed, most microorganisms adhering to the plant upper part of the rice which is a raw material will be sterilized.

Ethanol fermentation using the saccharified solution obtained by the saccharification method of the present invention can be performed using a microorganism that performs ethanol fermentation by assimilating saccharides having a low polymerization degree, particularly glucose, such as yeast and Zymomonas bacteria. it can. Specifically, it can be performed using yeast Saccharomyces cerevisiae (NBRC 0224).
Although it can be performed using microorganisms that have improved ethanol fermentation efficiency by genetic recombination technology such as recombinant Escherichia coli, focusing only on hexoses such as glucose and fructose, such as yeast and Zymomonas bacteria By using non-recombinant microorganisms, it is possible to reduce facility costs and management costs for measures for preventing the spread of genetically modified microorganisms.

Ethanol fermentation using the saccharified solution obtained by the saccharification method of the present invention can be performed using a known fermentation method.
In the present invention, the saccharification treatment step and the fermentation step can be separated, and the obtained sugar solution can be transferred to a batch-type or continuous fermentation tank. “Parallel double fermentation” is particularly desirable in terms of effects such as space saving and shortening of processing time.

The concentration of saccharides in the saccharified solution used for the ethanol fermentation is in the range of about 0.5% to 30%, preferably in the range of 5% to 30%.
The higher the saccharide concentration of the saccharified solution, the better the fermentation efficiency and the distillation efficiency, but when the operability is taken into consideration, the above range is preferable. For example, when a lignocellulosic raw material is suspended, it becomes bulky and the operability such as stirring is lowered.
Moreover, when the saccharide | sugar density | concentration of a saccharified liquid is lower than about 0.5% (preferably 5%), sufficient ethanol amount cannot be produced and it is undesirable.
In addition, when performing parallel double fermentation in which saccharification reaction and ethanol fermentation are performed simultaneously, the amount of rice straw as a raw material is adjusted so that the concentration of sugars expected to be produced falls within the predetermined range. Should. For example, when production of 0.25 gram of glucose from 1 gram of dry raw material (dry rice straw) can be expected by the saccharification method in the present invention, 2% or more of the dry raw material (2% or more with respect to the reaction solution for parallel double fermentation) By using dried rice straw, it is expected that 0.5% or more of glucose is produced.

Although the reaction temperature in the said ethanol fermentation changes with microorganisms to be used, in the case of yeast, it is the range of 25 to 50 degreeC, and in the range of 25 to 60 degreeC in bacteria. The reaction time largely depends on the yeast cell concentration, the amount of saccharifying enzyme, the reaction temperature, the stirring speed, etc., but in the batch method, about 10 to 48 hours is appropriate.
Moreover, when parallel double fermentation which performs saccharification reaction and ethanol fermentation simultaneously is performed, the saccharification enzyme and yeast contained in a liquid are moved by moving the liquid used for parallel double fermentation to the fermentor containing the following raw material after fermentation. Can be made to act again on the next raw material as it is.

As described above, according to the saccharification method of the present invention, there is no problem of cost and environmental load associated with the use of chemicals, remarkably high saccharification only by easy operation, in particular, production of glucose. It has become possible to dispose of rice plants that have not been used or have not been fully utilized until now (ie, rice straw), specifically, rice that does not eat mature fruits (rice). It is possible to construct a system that can be used as a composite biomass such as the above-ground part of a variety lineage plant, the above-ground part of a plant body excluding the matured fruit (rice) harvested during rice harvesting, and a raw material for ethanol production.
In particular, as a raw material for the saccharification method of the present invention, when using the above-ground part of the heading rice plant that does not eat mature fruits (rice), the cultivation period until the mature fruits (rice) are cut (recovered) Compared to, the cultivation period can be significantly shortened, and more than two cropping crops can be implemented throughout Japan, increasing the annual yield. In addition, matured rice with higher starch content It is considered that the applicability of the technique of the present invention is drastically improved when the leaves and leaves (rice straw) excluding the fruit are obtained in the late maturity stage.

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

Example 1
The leaf sheath and straw (rice straw) from which the cuttings were cut at the ground of rice (variety name: Habataki) were dried at 70 ° C. for 72 hours, and then pulverized (CMT Science High Speed Vibration Sample Crusher TI-) 100) for 90 seconds to prepare rice straw powder. The starch ratio (analyzed by Total starch kit (Megazyme)) and the total glucan ratio (converted as the amount of glucose after sulfuric acid treatment as glucan) per dry weight of the rice straw stems and leaves were 23% and 44.6%, respectively. was.
Two of this were prepared by weighing 100 mg of rice straw powder and putting it in a 2 ml plastic tube. 0.5 ml of water was added to these, and one of them (Sample 1-1) was heat-treated in a heat block at 100 ° C. for 10 minutes (stirring every 2 minutes).
Then, cellulase preparation (0.01 ml, Celluclast 1.5 L, Novozymes Japan), hemicellulase preparation (0.01 ml, Viscozyme L, Novozymes Japan), β-glucosidase preparation (0.003 ml, Novozyme188, Sigma) ) And amyloglucosidase (2 mg, 92 U / mg, Oriental Yeast Co., Ltd.) and sodium acetate buffer solution (final concentration of reaction solution 50 mM, pH 5.0) to make up to 1 ml to make reaction solution, heat at 50 ° C. The saccharification treatment was performed while rotating in the block.
In the middle, after sampling a part and diluting with water, the amount of free glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.). As a result, as shown in FIG. 1, about 70% of the theoretical amount of glucose was liberated in both samples after 4 hours of reaction.

  As a result, the rate was higher for the sample treated at 100 ° C. (Sample 1-1), but almost constant at a conversion rate similar to that of the sample not treated at 100 ° C. (Sample 1-2). became.

Example 2
50 mg of rice straw powder (variety name: Habataki) used in Example 1 was weighed. Two of these were put in a 2 ml plastic tube, 0.5 ml of water was added, and heat treatment was carried out for 10 minutes in a heat block at 100 ° C. (stirring every 2 minutes).
After that, one of them (Sample 2-1) includes cellulase preparation (Celluclast 1.5 L, 0.01 ml, Novozymes Japan), hemicellulase preparation (Viscozyme L, 0.01 ml, Novozymes Japan), β-glucosidase preparation. (0.003 ml, Novozyme188, Sigma) and amyloglucosidase (2 mg, 92 U / mg, Oriental Yeast Co., Ltd.) and sodium acetate buffer (final concentration of reaction solution 50 mM, pH 5.0) were added.
The other two (sample 2-2) include the latter two enzymes (β-glucosidase preparation (0.003 ml, Novozyme188, Sigma) and amyloglucosidase (2 mg, 92 U / mg, Oriental Yeast Co., Ltd.)) Sodium acetate buffer (final concentration of reaction solution 50 mM, pH 5.0) was added.
And each was made up to 1 ml to make a reaction solution, and saccharification was performed while rotating at 50 ° C. On the way, after sampling a part and diluting with water, the amount of free glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.).

  As a result, as shown in FIG. 2, glucose was liberated in both samples, but in Sample 2-2, about 34% of the theoretical amount of glucan was converted to glucose, whereas in Sample 2-1, About 67% of the theoretical amount of glucan was converted to glucose by the action of cellulose or hemicellulose-degrading enzyme system.

Example 3
The leaf sheath and straw (rice straw) from which the harvested ears were cut off at the border of rice (variety name: Takanari) were dried at 70 ° C. for 72 hours and then used with a grinder (Wiley grinder, 3 mm mesh pass). And pulverized to prepare rice straw powder. The starch ratio (analyzed by Total starch kit (Megazyme)) and the total glucan ratio (converted as the amount of glucose after sulfuric acid treatment as glucan) per dry weight of the rice straw stems and leaves were 9.79% and 38. It was 7%. 50 mg of this was weighed into a 2 ml plastic tube and used as sample 3-1.
In addition, the leaf sheath and straw (rice straw), except for the ears after harvesting rice, harvested with a normal rice harvester for rice (variety name: Milky Queen), were coarsely crushed and then crushed with a hammer mill (Yoshida Seisakusho). A straw powder was prepared. The starch ratio (analyzed by Total starch kit (Megazyme)) and the total glucan ratio (converted as the amount of glucose after sulfuric acid treatment as glucan) per dry weight of the rice straw stems and leaves were 2.18% and 34. It was 0%. 50 mg of this was weighed into a 2 ml plastic tube and used as sample 3-2.
0.5 ml of water was added to these and heat-treated in a heat block at 100 ° C. for 10 minutes (stirring every 2 minutes).
Cellulase preparation (0.01 ml, Celluclast 1.5 L, Novozymes Japan), hemicellulase preparation (0.01 ml, Viscozyme L, Novozymes Japan), β-glucosidase preparation (0.003 ml, Novozyme188, Sigma) And amyloglucosidase (2 mg, 92 U / mg, Oriental Yeast Co., Ltd.) and sodium acetate buffer (final concentration of reaction solution 50 mM, pH 5.0), and each made up to 1 ml was used as a reaction solution. The saccharification treatment was performed while rotating at 0 ° C.
On the way, after sampling a part and diluting with water, the amount of free glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.).

  As a result, as shown in FIG. 3, glucose was liberated in both samples, but 12.2 mg of glucose was liberated in sample 3-1, whereas it remained at 4.39 mg in sample 3-2.

  In addition, the same operation as that of Sample 3-1 was performed using samples in which the heat treatment temperature for 10 minutes before the enzyme treatment was changed to four types of 60 ° C., 70 ° C., 80 ° C. and no heat treatment, and the temperature of the heat treatment And the relationship between the amount of glucose released 4 hours after the enzyme reaction. The results are shown in Table 1.

Example 4
The leaf sheath and straw (rice straw) from which the harvested ears were cut off at the border of rice (variety name: Takanari) were dried at 70 ° C. for 72 hours and then used with a grinder (Wiley grinder, 3 mm mesh pass). And pulverized to prepare rice straw powder. The starch ratio (analyzed by Total starch kit (Megazyme)) and the total glucan ratio (converted as the amount of glucose after sulfuric acid treatment as glucan) per dry weight of the rice straw stems and leaves were 9.79% and 38. It was 7%.
Four of this were prepared by weighing 50 mg into a 2 ml plastic tube, added with 0.5 ml of water, and heat-treated in a heat block at 100 ° C. for 10 minutes (stirring every 2 minutes).
Thereafter, cellulase 1 (cellobiohydrolase I, 0.7 U / ml, Megazyme), cellulase 2 (endoglucanase II, 540 U / ml, Megazyme), each of four (Sample 4-1 to Sample 4-4) ) Was added as follows.
That is, sample 4-3 has cellulase 1 = 0 ml and cellulase 2 = 0 ml, and sample 4-2 has cellulase 1 = 0.1 ml and cellulase 2 = 0 ml, sample 4-3. Was added so that cellulase 1 = 0 ml and cellulase 2 = 0.1 ml, and to sample 4-4 so that cellulase 1 = 0.05 ml and cellulase 2 = 0.05 ml.
For each sample, β-glucosidase preparation (0.003 ml, Novozyme188, Sigma) and amyloglucosidase (2 mg, 92 U / mg, Oriental Yeast Co., Ltd.) and sodium acetate buffer (final concentration of reaction solution 50 mM) , PH 5.0) was added to make up each to 1 ml, and a reaction solution was used, and saccharification treatment was performed while rotating at 50 ° C.
On the way, after sampling a part and diluting with water, the amount of free glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.).

  As a result, as shown in FIG. 4, glucose was released in each sample. In Sample 4-1, about 6 mg of glucose was released after 4 hours, whereas in Sample 4-2, it was slightly released. In Sample 4-3, the amount of glucose released increased relatively remarkably. And about sample 4-4, the synergistic effect of cellulase 1 (cellobiohydrolase I) and cellulase 2 (endoglucanase II) was confirmed.

Example 5
Each of the samples shown in Table 2 (including those tested under a plurality of cultivation conditions for the same variety line) was cut off at the ground at the time indicated in Table 2, and the leaf sheath and cocoon were cut at 70 ° C. Each rice straw powder was prepared by drying for 72 hours and pulverizing for 90 seconds using a pulverizer (CMT Scientific High-Speed Vibration Sample Crusher TI-100). The starch rate per dry weight (analyzed by Total starch kit (Megazyme)) and the total glucan rate (the amount of glucose after sulfuric acid treatment was converted as glucan) were measured. (Measurement results are shown in Table 2.)
50 mg of these were weighed out and prepared in a 2 ml plastic tube, and 0.5 ml of water was added thereto, followed by heat treatment for 10 minutes in a heat block at 100 ° C. (stirring every 2 minutes).
Cellulase preparation (0.01 ml, Celluclast 1.5 L, Novozymes Japan), hemicellulase preparation (0.01 ml, Viscozyme L, Novozymes Japan), β-glucosidase preparation (0.003 ml, Novozyme188, Sigma) And amyloglucosidase (2 mg, 92 U / mg, Oriental Yeast Co., Ltd.) and sodium acetate buffer (final concentration of reaction solution 50 mM, pH 5.0) were added to make up 1 ml of each reaction solution. The saccharification treatment was carried out while rotating.
Then, after a part was sampled after 4 hours and diluted with water, the amount of free glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.). The measurement results are shown in Table 2.

  As a result, it was confirmed that the higher the starch content, the lower the non-starch glucan content, and the higher the starch content, the higher the saccharification rate of the glucan after the enzyme reaction.

Example 6
The leaf sheath and straw (rice straw) from which the cuttings were cut off at the border of rice (variety name: Habataki) were dried at 70 ° C. for 72 hours, and then pulverized (CMT Science High-Speed Vibration Sample Crusher TI-) 100) for 90 seconds to prepare rice straw powder a. The starch ratio (analyzed by Total starch kit (Megazyme)) and the total glucan ratio (converted as the amount of glucose after sulfuric acid treatment as glucan) per dry weight of the rice straw stems and leaves were 24% and 44.6%, respectively. was.
25 mg of each of these was weighed out and placed in a 2 ml plastic tube to prepare Sample 6-1 and Sample 6-2.
In addition, rice straw including leaf sheaths and straw except for harvested rice harvested with a conventional rice mower of rice (variety name: Koshihikari) was coarsely crushed and then crushed with a hammer mill (Yoshida Seisakusho). Powder b was prepared. The starch ratio (analyzed by Total starch kit (Megazyme)) and the total glucan ratio (converted as the amount of glucose after sulfuric acid treatment as glucan) per dry weight of the rice straw stems and leaves were 2.77% and 30. respectively. It was 9%.
25 mg of this was weighed out and put into a 2 ml plastic tube to prepare a sample 6-3. Then, 0.5 ml of water was added to each of Sample 6-1, Sample 6-2, and Sample 6-3, and heat-treated in a heat block at 100 ° C. for 10 minutes (stirring every 2 minutes).

Next, sample 6-1 (rice straw powder a) and sample 6-3 (rice straw powder b) were charged with 0.05 ml of cellulase 1 (cellobiohydrolase I, 0.7 U / ml, Megazyme) and 0.05 ml cellulase 2 (Endoglucanase II, 540 U / ml, Megazyme), 0.025 ml β-glucosidase (40 U / ml, Megazyme), 2 mg amyloglucosidase (92 U / mg, Oriental Yeast Co.) and 0.004 ml of lichenase (1000 U / ml, Megazyme) was added.
Sample 6-2 (rice straw powder a) was added with the same amounts of cellulase 1, cellulase 2, and β-glucosidase as described above.
To these, a sodium acetate buffer solution (final concentration of reaction solution 50 mM, pH 5.0) was added, and each was made up to 1 ml. The reaction solution was used, and a saccharification reaction was performed while rotating at 50 ° C. On the way, after sampling a part and diluting with water, the amount of free glucose was measured using Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.).

As a result, as shown in FIG. 5, 6.6 mg or more of glucose was liberated in the sample 6-1 after 4 hours of reaction, 2.37 mg in the sample 6-2, and 1.46 mg in the sample 6-3. .
When samples 6-1 and 6-2 were compared, it was revealed that sample 6-1 released more glucose. Sample 6-2 is decomposed only with β-glucan-degrading enzyme, and sample 6-1 is decomposed assuming both α-glucan and β-glucan.
Moreover, the result that the decomposition efficiency of β-glucan of sample 6-2 is higher than that of sample 6-3 indicates that the β-glucan structure of rice straw at the heading stage containing starch at a high concentration Compared to the β-glucan structure of mature rice straw, which contains only low concentrations, it was more susceptible to enzymatic degradation.

Example 7
25.0 mg of the rice straw powder raw material of Sample 5-15 (Habataki, heading period) of Example 5 was weighed and prepared in two 2 ml plastic tubes. To each of these, 0.45 ml of YEP medium (containing 2% Yeast Extract and 1% Bacto Peptone and adjusted to pH 5.0) was added and left on a heat block at 100 ° C. for 10 minutes. The mixture was stirred for about 3 seconds by a vortex mixer every minute.
One of them includes cellulase preparation (0.01 ml, Celluclast 1.5 L, Novozymes Japan), hemicellulase preparation (0.01 ml, Viscozyme L, Novozymes Japan), β-glucosidase preparation (0.003 ml, Novozyme 188 (Sigma) and amyloglucosidase (2 mg, 92 U / mg, Oriental Yeast Co., Ltd.) containing 0.5 ml of YEP medium was prepared as Sample 7-1. The other was prepared by adding only 0.5 ml of YEP medium as sample 7-2.
Samples having the same liquid composition as Samples 7-1 and 7-2 and not containing rice straw powder raw materials were prepared as Sample 7-3 and Sample 7-4, respectively.
Next, Saccharomyces cerevisiae (NBRC 0224) was inoculated in advance in YPPG medium (containing 2% Yeast Extract, 1% Bacto Peptone and 1% glucose and adjusted to pH 5.0) and rotated at 30 ° C. for 5 hours. The cell suspension after (24 rotations / min) was added to each sample in an amount of 0.05 ml, and after closing, the cells were similarly cultured for 15 hours.
After the culture, the amount of ethanol contained in the culture supernatant was quantified using Ethanol Assay Kit (manufactured by Megazyme) and calculated as the ethanol content per ml of liquid. The measurement was repeated twice. The results are shown in Table 3.

As shown in Sample 7-1, it was shown that ethanol was produced by simultaneously performing saccharification reaction and ethanol fermentation (parallel double fermentation) in the presence of rice straw powder (saccharification raw material).
Moreover, in the sample 7-2 to which only the rice straw powder (saccharification raw material) and the sample 7-4 having only the YEP medium, which are samples in which the saccharification reaction does not proceed, ethanol production is extremely low, and ethanol fermentation is almost performed. There wasn't.
It was observed that ethanol fermentation also occurred in Sample 7-3, which is a sample in which saccharification reaction does not proceed, and in which rice straw powder (saccharification raw material) does not exist, but saccharides contained in some enzyme preparations are mainly used. It is considered to be a fermentation source.
From these results, it was shown that ethanol was produced by performing ethanol fermentation with yeast using a saccharified solution obtained by saccharifying rice straw powder.

Test example 1
In order to prepare each sample shown in Table 4, after cutting the above-ground part of the plant body of the rice from the ground at the time (maturity or heading) described in Table 4, after removing the ears and taking out the leaf sheath and straw, “Dried for 72 hours at 70 ° C. within 24 hours (heat drying treatment)”. Then, it grind | pulverized for 90 second using the grinder (CMT science high-speed vibration sample grinder TI-100), and prepared the rice straw powder (Sample E1-1-Sample E1-13, Sample E1-15).
Next, these samples were extracted with water, and the total fructose amount (the total amount obtained by converting the free fructose amount and the fructose residue in the sucrose molecule as free fructose) was measured by the Roe method.
In addition, the amount of free glucose (amount not containing glucose residues in sucrose molecules) was measured by Glucose C-II Test Wako (Wako Pure Chemical Industries, Ltd.).
As a comparative control, the above-ground part of the mature rice plant (Koshihikari, indicated by * in Table 4) was cut off from the ground, the ears were removed and the leaf sheath and cocoon were taken out, and then stored at room temperature for two weeks. The rice straw powder (sample E1-14), which was coarsely pulverized and then pulverized with a hammer mill (manufactured by Yoshida Seisakusho), was measured in the same manner as described above.
Then, using the above formulas (I) to (III), the total amount of free carbohydrates contained in the sample (the amount of sucrose and the amount of free glucose) from the values of the total fructose amount and free glucose amount measured as described above And the sum of the amount of free fructose). The results are shown in Table 4.

  As a result, the above-ground part of the plant body of rice is cut and dried at 70 ° C. for 72 hours within 24 hours (by performing heat drying treatment). It was shown that carbohydrates are retained at high concentrations.

Test example 2
The above-ground part of the mature rice plant (milky queen) was cut with a normal harvesting machine, and after removing the ears, the leaf sheath and the cocoon were taken out, and “air-dried indoors within 24 hours (drying treatment)”. Then, coarsely pulverized and further pulverized using a hammer mill (manufactured by Yoshida Seisakusho) to prepare a rice straw powder (sample E2-1) having a water content of 5%.
Next, 10 mg of rice straw powder of sample E2-1 was weighed into a 2 ml plastic tube, and rice straw powder (sample E2-2) stored at room temperature (about 25 ° C.) for 72 hours after closing was prepared. . That is, it was stored at room temperature in a dry state.
In addition, 10 mg of rice straw powder of this sample E2-1 was weighed into a 2 ml plastic tube, sterilized water was added to obtain rice straw powder having a water content of 81%, and then closed and room temperature (about 25 ° C.) The rice straw powder (sample E2-3) stored for 24 hours and the rice straw powder (sample E2-4) stored for 48 hours were prepared. That is, it was stored at room temperature with a high water content.
In addition, 10 mg of rice straw powder of this sample E2-1 was weighed into a 2 ml plastic tube, sterilized water was added to obtain rice straw powder having a moisture content of 81%, and then sealed and stored at 65 ° C. for 4 hours. Furthermore, rice straw powder (sample E2-5) stored for 44 hours at room temperature (about 25 ° C.) was prepared. That is, heat treatment was performed immediately after the moisture content was high, and then the mixture was stored at room temperature with a high moisture content.
And about the rice straw powder of these sample E2-1-sample E2-5, it carried out similarly to the method of the test example 1, and measured the total fructose amount by Roe method. The results are shown in Table 5.

As Table 5 shows, the total fructose content (free saccharides) contained in rice straw powder decreased rapidly when stored at room temperature with a high water content.
On the other hand, in the case where the moisture content is increased and then immediately heated at 65 ° C. and then stored at room temperature with a high moisture content, the total fructose amount (free carbohydrate) is measured before storage at room temperature (before the test). And almost the same value.
In addition, the total amount of fructose (free carbohydrate) hardly changed in the case where the water content was kept at a low temperature (dry state) and stored at room temperature.
In this way, free sugars in rice straw powder rapidly decrease when left at room temperature after humidification. In order to retain the free sugar in the raw material, heat treatment or drying after the drying process is performed. Saving in a state has been shown to be effective.

Test example 3
Rice straw powder prepared in Test Example 1 (Samples E1-1, E1-2, E1-5 to E1-7, E1-9, E1-10, E1-12: all above the plant part above the mature stage) Rice-powder, rice straw powder from which leaf sheath and cocoon were taken out except for the ears) β- (1 → 3), β- (1 → 4) glucan content in Mixed-Linkage Beta -Measured using Glucan kit (Megazyme).
Moreover, these starch content was also measured using Total starch kit (Megazyme), and the starch ratio per dry weight of the foliage was determined. The results are shown in Table 6.

As a result, the above-ground part of rice plants having a starch rate of 5% or more (samples E1-1, E1-2, and E1-5) has the above-ground part of rice plants having a starch rate lower than 5% (sample E1- 6, E1-7, E1-9, E1-10 and E1-12), β- (1 → 3), β- (1 → 4) glucan content tends to be higher. It was.

The present invention relates to an efficient biomass saccharification technology that uses low-cost, low-environmental environmentally-friendly biomass saccharification technology, which uses raw material of rice plant containing 5% or more of starch per dry weight of foliage. It is expected to lead to the development of manufacturing technology and biorefinery technology.
In particular, it is recognized as extremely important as a new innovation in the development of bioethanol production technology, which is an urgent issue not only in Japan but around the world.
Further, according to the present invention, by-products such as enzyme saccharification residue can be used in feed, fertilizer and fuel fields.

In Example 1, it is a figure which shows the time-dependent change of the glucan hydrolysis degree in the saccharification process of rice straw powder. Black squares in the graph indicate Sample 1-1, and black diamonds indicate Sample 1-2. It is a figure which shows the time-dependent change of the glucan hydrolysis degree in the saccharification process of the rice straw powder in Example 2. In the graph, a black diamond indicates Sample 2-1, and a black rectangle indicates Sample 2-2. It is a figure which shows the time-dependent change of the glucose release amount in the saccharification process of the rice straw powder in Example 3. In the graph, a black diamond represents the sample 3-1, and a black square represents the sample 3-2. It is a figure which shows the time-dependent change of the glucose release amount in the saccharification process of the rice straw powder in Example 4. In the graph, black diamonds indicate Sample 4-1, black squares indicate Sample 4-2, black triangles indicate Sample 4-3, and crosses indicate Sample 4-4. It is a figure which shows the time-dependent change of the glucose release amount in the saccharification process of the rice straw powder in Example 6. In the graph, a black diamond represents the sample 6-1, the black square represents the sample 6-2, and the triangle represents the sample 6-3.

Claims (13)

  The plant body part of rice plants containing 5% or more starch per dry weight in the foliage is collected, ground, and then saccharified using an enzyme solution containing amyloglucosidase and β-glucan degrading enzyme. Saccharification method.   The saccharification method according to claim 1, wherein the above-ground part of the plant body is a foliage part excluding mature fruits.   The saccharification method according to claim 1, wherein the above-ground part of the plant body is from the heading stage to the mature stage.   The saccharification method according to any one of claims 1 to 3, wherein the above-ground part of the plant body includes a border part. After recovering the above-ground part of the plant body, by performing a heat treatment at 40 to 130 ° C. and / or performing a drying treatment within 24 hours, the above-ground part of the plant body is converted into the above-mentioned part of the plant body. Sucrose, glucose and fructose utilization activity of the plant body cells and microorganisms that inhabit the plant body and the surface thereof are suppressed,
The saccharification method according to any one of claims 1 to 4.   6. The method according to claim 5, wherein the above-ground part of the plant body in which the assimilation activity is suppressed is 4% or more of sucrose and 0. 0% of glucose with respect to the dry weight of the above-ground part of the plant body. The saccharification method according to claim 5, wherein the saccharification method comprises 5% or more and 0.5% or more of fructose.   The saccharification method in any one of Claims 1-6 which collect | recovers 50% or more as glucose among the total glucan content contained in the said plant body above-ground part.   The β-glucan degrading enzyme has an activity of hydrolyzing at least one of amorphous cellulose, crystalline cellulose, and β- (1 → 3), β- (1 → 4) -glucan. The saccharification method in any one of Claims 1-7 which is a thing.   The saccharification method according to any one of claims 1 to 8, wherein the enzyme solution containing amyloglucosidase and β-glucan degrading enzyme has both cellobiohydrolase and endoglucanase activities.   The saccharification method according to any one of claims 1 to 9, wherein the enzyme solution containing amyloglucosidase and β-glucan-degrading enzyme contains at least one of hemicellulose-degrading enzyme, β-glucosidase, and α-amylase. .   The saccharification method according to any one of claims 1 to 10, wherein the amyloglucosidase and / or β-glucan degrading enzyme is a microorganism-derived enzyme or enzyme preparation.   The saccharification method according to any one of claims 1 to 11, wherein a heat treatment at 70 to 130 ° C is performed before the saccharification treatment and / or in the saccharification treatment.   A method for producing ethanol, comprising performing ethanol fermentation using the saccharified solution obtained by the saccharification method according to any one of claims 1 to 12.

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