JP2015037389A - Improved method of enzyme saccharifying reaction from lignocellulose biomass by supplementation of a lipolytic enzyme and nonionic surfactant and simultaneous saccharifying fermentation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technology for reducing cost for saccharization in which the saccharifying reaction is proceeded with a smaller amount of supplementation of cellulase and a larger amount of saccharide is yielded in order to solve a conventional problem that cellulase requires the maximum cost in the saccharifying reaction, since the cellulase is absorbed in a non specific manner in a part other than lignocellulose biomass and an active part just after supplementation of the cellulase into an enzyme saccharifying reaction container, and further amount of cellulase is used in saccharifying process of lignocellulose biomass due to an effect from a cellulase activity inhibitor derived from biomass eluted upon hydrolysis reaction.SOLUTION: The inventors of the present application made the present invention by intensive research for solving the problem. They found that efficiency in saccharization of lignocellulose biomass was improved by supplementation of a lipolytic enzyme or supplementation of the lipolytic enzyme and nonionic surfactant upon proceeding enzymic saccharization of the lignocellulose biomass and that inhibition in fermentation is avoided by proceeding simultaneous saccharifying fermentation under the presence of the lipolytic enzyme and the nonionic surfactant.

Description

  The technical field of the present invention relates to a method for producing sugar from lignocellulosic biomass by enzymatic saccharification and a method for producing ethanol by simultaneous saccharification and fermentation from lignocellulosic biomass.

  Japan is a lush country where forests account for approximately 70% of the country, and in addition to cedar, cypress and pine, there are abundant unused lignocellulosic biomass such as rice straw and straw. Such unused lignocellulosic biomass is the most important on earth not only for the production of biomass ethanol from non-food resources but also for the production of sugar as a raw material for food, chemical industry and fermentation after the depletion of petroleum resources. A good carbon source (Non-patent Document 1).

  However, lignocellulosic biomass that exists in nature is a complex composition of cellulose, hemicellulose, and lignin. A pretreatment for destroying the structure of lignin, cellulose, and hemicellulose by the treatment is performed (Non-patent Document 2), or a plurality of the above-mentioned pretreatments are combined. Despite these pretreatments, lignocellulosic biomass enzymatic saccharified liquid has not reached a level that can be practically used in terms of price and fermentation efficiency in conjunction with subsequent ethanol fermentation, etc. References 3, 4).

This is due to the fact that the activity of β-glucosidase is inhibited by cellulose-derived glucose, cellobiose and hemicellulose-derived mannose, galactose, and xylose (non-patent document 5) as well as xylan and soluble starch. In addition to polysaccharides such as pectin, carbohydrates such as xylose, arabinose, and glucuronic acid, furfural, vanillin, syringaldehyde, trans-cinnamic acid, and 4-hydroxybenzoic acid inhibit cellulase and β-glucosidase activities. (Non-Patent Document 6) is known. Substances such as those mentioned above gradually elute from lignocellulosic biomass during the hydrolysis reaction and inhibit the activity of cellulase. In addition to the addition of cellulase to the enzymatic saccharification vessel, This is because expensive cellulase is used more than necessary by nonspecific binding (Non-patent Document 7). In addition, lignocellulosic biomass contains a maximum of about 4% oil (Non-patent Document 8), and this oil covers the surface of the biomass by pretreatment, so that the biomass used as cellulase and substrate It is expected that the enzymatic saccharification reaction has not been sufficiently performed.
Furthermore, since the obtained saccharified solution contains a large amount of fermentation inhibiting substances, it cannot be put into practical use as it is, and it is necessary to remove the fermentation inhibiting substances contained in the saccharified solution after the saccharification reaction. I have it.

JP 2010-246422 Sera Yutaka et al.: Simultaneous saccharification and fermentation of cellulosic materials JP 2012-254072 Masamasa et al: Method for producing ethanol from lignocellulose-containing biomass Naoyuki Okuda et al.: Method for producing ethanol Japanese Patent Application No. 2012-235103 Hoshino et al.: A method for producing sugar via a step of removing an enzyme inhibitor Japanese Patent Application No. 2013-064750 Hoshino et al.: Ethanol production method using Antarctic yeast at low temperature

J. Nowak et al .: Composite structure of wood cells in petrified wood. Materials Science and Engineering: C, 25, 119-130, 2005 Y. Yamashita et al: Effective enzyme saccharification and ethanol production from Japanese cedar using various pretreatment methods. Journal of Bioscience and Bioengineering, 110, 79-86, 2010 Kosuke Kimoto: Bioethanol production process from wood-based raw materials. Mitsui Engineering & Shipbuilding Technical Report, 201, 24-29, 2010 Kenji Asano et al.: Bioethanol production cost and CO2 reduction cost analysis in Japan. [Online] AIST / BTRC DISCUSSION PAPER, July, 2007, Internet <https://unit.aist.go.jp/btrc/research_result/documents/ DISCUSSIONPAPER.pdf> Z. Xiao et al .: Effect of Sugar Inhibition on Cellulases and β- Glucosidase During Enzymatic Hydrolysis of Softwood Substrates. Applied Biochemistry and Biotechnology. 113-116, 1115-1126, 2004 E. Ximenes et al .: Inhibitor of cellulases by phenols. Enzyme and Microbial Technology, 46, 170-176, 2010 T. Eriksson et al .: Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose, Enzyme and Microbial Technology, 31, 353-364, 2002 P Gupta et al: Tissue culture of forest trees *--Clonal propagation of mature trees of Eucalyptus citriodora Hook, by tissue culture Plant Science Letters 20, 195-201, 1981

  Under the above-mentioned circumstances, the present invention provides (1) reducing the amount of cellulase used to hydrolyze lignocellulosic biomass without going through complicated steps, and obtaining more sugar, or (2) An object of the present invention is to provide a method for converting lignocellulosic biomass in order to obtain a large amount of ethanol by adding fermentable microorganisms during hydrolysis and performing simultaneous saccharification and fermentation.

  In order to achieve the above-mentioned object, the inventors of the present invention efficiently made a saccharified solution by adding a lipolytic enzyme or a lipolytic enzyme and a nonionic surfactant at the start of a saccharification reaction of lignocellulosic biomass. I found that I can get well. Furthermore, when adding cellulase, lipolytic enzyme, and nonionic surfactant to lignocellulosic biomass, the present inventors can simultaneously produce saccharification and fermentation to add fermentable microorganisms and efficiently produce ethanol. I found it. Thus, the present inventors have completed the present invention, which is a biomass conversion method characterized by adding a lipolytic enzyme or a lipolytic enzyme and a nonionic surfactant to a saccharification reaction system or a simultaneous saccharification and fermentation system. I let you.

  The saccharified solution obtained by adding the lipolytic enzyme of the present invention and a nonionic surfactant had a glucose concentration about twice that of the conventional saccharified solution.

  According to the present invention, by adding a lipolytic enzyme or a lipolytic enzyme and a nonionic surfactant during lignocellulosic biomass hydrolysis reaction, the amount of cellulase added is 1/5 or less than when performing a normal enzyme saccharification reaction Can be. In addition, by adding lipolytic enzymes and nonionic surfactants during simultaneous saccharification and fermentation, approximately 2.5 times the amount of ethanol can be produced compared to conventional simultaneous saccharification and fermentation. , Extremely effective.

The glucose concentration (●) when the enzymatic saccharification reaction was carried out under the conditions of Example 1. The glucose concentration (●) when the enzymatic saccharification reaction was carried out under the conditions of Example 2. The glucose concentration (●) when the enzymatic saccharification reaction was carried out under the conditions of Comparative Example 1. The glucose concentration (●) when the enzymatic saccharification reaction was carried out under the conditions of Comparative Example 2. The glucose concentration (▲) and ethanol concentration (●) when performing simultaneous saccharification and fermentation with Mrakia blollopis performed under the conditions of Example 3. The glucose concentration (▲) and ethanol concentration (●) when simultaneous saccharification and fermentation using Mrakia blollopis performed under the conditions of Comparative Example 3 were performed.

1. 1. Introduction As described above, the inventors of the present invention can prevent a decrease in cellulase activity by adding a lipolytic enzyme or a lipolytic enzyme and a nonionic surfactant to an enzymatic saccharification reaction by cellulase from lignocellulosic biomass. I found out.

The present invention includes the following methods.
(1) In the method for producing sugar from lignocellulosic biomass using an enzyme, the sugar yield is improved by adding a lipolytic enzyme to the enzymatic saccharification step.
(2) The method according to (1), wherein a nonionic surfactant is added in addition to the lipolytic enzyme.
(3) In the method of producing ethanol from lignocellulosic biomass by simultaneous saccharification and fermentation, the ethanol yield is improved by adding a lipolytic enzyme or lipolytic enzyme and a nonionic surfactant to the simultaneous saccharification and fermentation step. Method.
The raw material and reaction are demonstrated below.

2. Lignocellulosic biomass Lignocellulosic biomass that is the subject of the method of the present invention is obtained by converting a polysaccharide consisting of cellulose and hemicellulose into a tree by lignin, broad-leaved tree, conifer, bamboo grass, rice straw, rice husk, wheat straw, It may be lignocellulosic biomass with low or no lignin content, such as corn stover, other agricultural, forestry and fishery resources and their waste, wood fiber, wood chips, wood chips, pulp, waste paper, etc. Also includes cellulosic biomass and their waste. These biomass resources may be a combination of two or more.

Preferred lignocellulosic biomass is, for example, rice straw, wheat straw, eucalyptus wood flour, and cedar wood flour.
Eucalyptus wood flour is a representative material containing lignocellulose. The present invention is not limited to eucalyptus wood flour and can be applied to all biomass resources represented by lignocellulosic biomass. That is, the lignocellulosic biomass resource that is the subject of the present invention is a resource obtained by converting a structure of cellulose and hemicellulose into lignin. Conifers, hardwoods, sasa, bamboo, rice straw, rice husks, wheat straw, other agricultural and forestry resources and their waste, as well as wood fibers, wood chips and veneer scraps, pulps, waste paper, etc. is there. Biomass resources with low tannin / lignin content such as sugar resources such as sugar cane and sugar beet, and starch resources such as potato, sugar cane and sugar cane are also included. These biomass resources may be a combination of two or more.

2-1. Pretreatment of lignocellulosic biomass Lignocellulosic biomass can be obtained in the form of waste materials, etc., but in order to efficiently saccharify this, lignocellulose is usually used as an enzyme when pulverized and then dropped by an enzymatic method. The pre-processing which is decomposed by is performed.

  As the pretreatment in the present invention, any pretreatment method usually used in biomass saccharification may be employed. Examples of the pretreatment include (1) pulverization treatment, (2) steaming / explosion treatment or hydrothermal treatment, (3) dilute sulfuric acid treatment, (4) alkali treatment, (5) organosolv treatment, (6) biology Target pretreatment methods are known, and these can be combined. Preferably, mechanochemical treatment can be performed as pretreatment.

3. TECHNICAL FIELD The present invention relates to a method for producing sugar by enzymatic saccharification using an enzyme from lignocellulosic biomass. In the present invention, the enzymatic saccharification reaction of lignocellulosic biomass includes a saccharification process using lignocellulosic biomass with cellulase or a simultaneous saccharification and fermentation process using cellulase.

  Usually, as an enzyme used for enzyme treatment, hemicellulase can be included as needed in addition to cellulase. The saccharification conditions of biomass cellulase and / or hemicellulase can be based on the general conditions for conventional saccharification of biomass.

  The amount of cellulase added in the enzymatic saccharification step or the simultaneous saccharification and fermentation step (also referred to as a concurrent saccharification and fermentation step) is preferably in the range of 0.1 FPU to 200 FPU, more preferably in the range of 0.1 to 50 FPU, A cellulase addition amount in the range of ˜20 FPU is more preferred. When hemicellulase is also used, the amount of hemicellulase added to the enzymatic saccharification step or the simultaneous saccharification and fermentation step (also referred to as a concurrent saccharification and fermentation step) is preferably in the range of 0.1 U to 200 U per gram of dry substrate, 10 to 150 A hemicellulase addition amount in the range of U is more preferable, and a hemicellulase addition amount in the range of 20 to 100 U is more preferable.

  The pH in the enzymatic saccharification step or the simultaneous saccharification and fermentation step is preferably maintained in the range of 2.0 to 10.0, and more preferably in the range of 4.0 to 7.5.

  The temperature of the enzymatic saccharification step or the simultaneous saccharification and fermentation step is not particularly limited as long as it is within the optimum temperature range of the enzyme, preferably 0 to 50 ° C, more preferably 10 to 40 ° C.

  The reaction is preferably continuous, but may be semi-batch or batch. The reaction time varies depending on the enzyme concentration, but in the case of a batch system, it is 10 to 240 hours, more preferably 15 to 168 hours. Also in the case of the continuous type, the average residence time is 10 to 148 hours, more preferably 15 to 120 hours.

  In hydrolyzing cellulosic biomass in the present invention, the method and apparatus are not particularly limited, and known methods and apparatuses can be applied. Enzymatic saccharification using cellulase and hemicellulase can be applied to the hydrolysis method of cellulosic biomass, and a combination of enzymatic saccharification and acid saccharification may be used. The hydrolysis apparatus may be a batch type, a fixed bed reaction type, a continuous type, or the like.

  Alcohol fermentation in simultaneous saccharification and fermentation includes, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Schizosaccharomyces japonicas, Kluyveromyces lactis, Kluyveromyces marxianus, Scheffersomyces stipites, Candida sheyces, Candida sheyces It is possible to use yeasts such as the genus, Issatchenkia, Pichia, etc., alcohol-fermenting bacteria such as Zymomonas mobilis, Zymomonas, Zymobacter palmae, Zymobacter, and Clostrium.

  Further, the present simultaneous saccharification and fermentation can also use a yeast that acts at low temperature, specifically, the basidiomycetous fungus Agaricus phyllum fungus net cystofilobasidium cystophyllobidae Murchia, for example, Murakia brollopis strain SK-4 (Mrakia blollopis) (FERM P-22126 strain) can be mentioned as the most preferred strain.

4). Saccharification with cellulase and hemicellulase 4-1. Cellulose In the present specification, cellulose refers to a polymer in which glucose is polymerized by β-1,4 glucoside bonds and derivatives thereof. The degree of polymerization of glucose in cellulose is not particularly limited, but is preferably 200 or more. In addition, examples of the derivative include derivatives such as carboxymethylation, aldehyde formation, or esterification. Cellulose may also contain cellooligosaccharides and cellobiose, which are partially decomposed products. Further, the cellulose may be crystalline cellulose or non-crystalline cellulose, but preferably contains crystalline cellulose. Furthermore, the cellulose is not particularly limited as long as it is derived from biomass. It may be derived from plants, fungi, or bacteria.

4-2. Cellulase and hemicellulase Cellulase means a series of enzymes that break down cellulose into glucose. Cellulases are selected from these enzyme groups, but preferably endoglucanase (EC 3.2.1.74), cellobiohydrolase (EC 3.2.1.91), and β-glucosinase (EC 23.2.4.1, EC 3.2.1.21) Is mentioned.

  Although it does not specifically limit as a cellulase, It is preferable that it is a cellulase with high activity itself. Examples of such cellulases include Acremonium bacteria, Talaromyces bacteria, Trichoderuma reesei, Trichoderuma viride and other Trichoderma bacteria, Fusarium bacteria, Phanerochaete bacteria In addition to the genus Clostridium (Tremetes, Penicillium, Humicola, Aspergillus, Murakia, Mrakiella, etc. Clostridium bacteria, Pseudomonas bacteria, Cellulomonas bacteria, Ruminococcus bacteria, Bacillus bacteria, Sulfolobus bacteria, Streptomyces bacteria And cellulases derived from actinomycetes such as fungi and Thermoactinomyces. One or a combination of two or more of these cellulases can be used. Such cellulase may be artificially modified. Examples of the modification method include a point mutation method and mutation caused by irradiating a microorganism with ultraviolet rays.

  Any hemicellulase may be used as long as it is a hemicellulase conventionally used for saccharification of biomass. Hemicellulase is a general term for an enzyme group that catalyzes an enzyme reaction system that hydrolyzes hemicellulose into xylose, arabinose, mannose, galactose, and the like. There are enzymes called by various names such as xylosidase and mannosidase. Although it does not specifically limit as hemicellulase, It is preferable that it is hemicellulase with high activity itself. One or a combination of two or more of these various hemicellulases can be used.

  Such hemicellulases may also be artificially modified. Examples of the modification method include a point mutation method and mutation caused by irradiating a microorganism with ultraviolet rays.

  The saccharification reaction may be performed at a low temperature within the working temperature range of the enzyme, for example, within the working temperature range of the enzyme. For example, a range of 40 ° C. or lower can be employed.

4-3. Cellulase activity inhibitory substance The biomass-derived cellulase activity inhibitory substance means a substance that inhibits the activity of cellulase eluted when cellulosic biomass is enzymatically saccharified with cellulase. Specific examples include tannic acid, acetic acid, and gallic acid.

5. Simultaneous saccharification and fermentation of biomass As described above, it is known that the activity of cellulase is inhibited by the accumulation of glucose and cellobiose derived from cellulose, which is a product of the saccharification reaction by cellulase, in the reaction solution. In order to prevent this inhibition, saccharification and ethanol fermentation are performed simultaneously. Can be prevented. Such a method of simultaneously performing enzymatic saccharification treatment and ethanol fermentation is called a simultaneous saccharification and fermentation method.

  Also in this invention, saccharification from biomass and ethanol fermentation can also be performed simultaneously. In the present invention, there is no particular limitation on the method and apparatus for simultaneous saccharification and fermentation of biomass such as lignocellulosic biomass, and known methods and apparatuses can be applied. The simultaneous saccharification reaction apparatus may be a batch type, a fixed bed reaction type, a continuous type, or the like.

6). Lipolytic enzyme Here, the lipolytic enzyme refers to a group of enzymes that hydrolyze ester bonds constituting lipids. In particular, it refers to triacylglyceride lipase (EC 3.1.1.3) that decomposes triglycerides and releases fatty acids. Although it does not specifically limit as a lipolytic enzyme, It is preferable that it is a lipolytic enzyme with high activity itself. Examples of such lipolytic enzymes include Mrakia, Candida, Aspergillus, Cryptococcus, Fusarium, and Yarrowia. And lipolytic enzymes derived from fungi and bacteria such as Geotrichum, Typhula, Mrakiella, Clostridium, and Pseudomonas. One or a combination of two or more of these various lipolytic enzymes can be used. Such a lipolytic enzyme may be artificially modified.
Examples of the modification method include a point mutation method and mutation by irradiating a microorganism with ultraviolet rays, as in the case of cellulase.

7). Nonionic Surfactant Examples of the nonionic surfactant that can be used in the present invention include Tween 20, Tween 80, Triton X-100, Triton X-114, Brij35, Brij 58, and Digitonin.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

[Example 1]
Suspended in a hydrolysis reaction vessel containing 20 mM citrate buffer pH 5.0 so that the mecanomi chemical-treated eucalyptus wood flour was 20% (w / v) and heat-treated at 121 ° C. for 30 minutes. After cooling to room temperature, add Cellulase T “Amano” 4 (Amano Enzyme) 1.8 FPU / (g-substrate) and Optimash BG (Genencor) 20 μl / (g-substrate), followed by hydrolysis Lipidase lipase AS “Amano” was added to the solution at 5 U / (g-substrate) and stirred at 37 ° C. and 120 rpm for 120 hours. The hydrolyzate collected every 24 hours immediately after the start of the hydrolysis reaction was boiled for 3 minutes to completely deactivate the enzyme, and centrifuged at 15000 rpm for 10 minutes to recover the water-soluble part. The recovered water-soluble part was then analyzed by high performance liquid chromatography (HPLC) to determine the glucose concentration. FIG. 1 shows the glucose concentration in the hydrolysis test of Example 1. From the figure, a saccharified solution having a glucose concentration of about 42.8 g / l was obtained.

[Example 2]
Suspended in a hydrolysis reaction vessel containing 20 mM citrate buffer pH 5.0 so that the mecanomi chemical-treated eucalyptus wood flour was 20% (w / v) and heat-treated at 121 ° C. for 30 minutes. After cooling to room temperature, add Cellulase T “Amano” 4 (Amano Enzyme) 1.8 FPU / (g-substrate) and Optimash BG (Genencor) 20 μl / (g-substrate), followed by hydrolysis Add nonionic surfactant Tween 80 and 5 U / (g-substrate) lipolytic enzyme lipase AS “Amano” to 1% (v / v) of the solution, and 120 hours at 37 ° C. and 120 rpm Stirring was performed. The hydrolyzate collected every 24 hours immediately after the start of the hydrolysis reaction was boiled for 3 minutes to completely deactivate the enzyme, and centrifuged at 15000 rpm for 10 minutes to recover the water-soluble part. The recovered water-soluble part was then analyzed by high performance liquid chromatography (HPLC) to determine the glucose concentration. FIG. 2 shows the glucose concentration in the hydrolysis test of Example 2. From the figure, a saccharified solution having a glucose concentration of about 47.5 g / l was obtained.

[Comparative Example 1]
20 mM citrate buffer (pH 5.0) was added to mecanomi chemical-treated eucalyptus wood flour in a hydrolysis reaction vessel and heated at 121 ° C. for 30 minutes. After cooling to room temperature, add Cellulase T “Amano” 4 (Amano Enzyme) 9 FPU / (g-substrate) and Optimash BG (Genencor) 20 μl / (g-substrate), 37 ° C., 120 rpm For 120 hours. The hydrolyzate collected every 24 hours immediately after the start of the hydrolysis reaction was boiled for 3 minutes to completely deactivate the enzyme, and centrifuged at 15000 rpm for 10 minutes to recover the water-soluble part. The recovered water-soluble part was then analyzed by high performance liquid chromatography (HPLC) to determine the glucose concentration. FIG. 3 shows the glucose concentration in the hydrolysis test of Comparative Example 1. From the figure, a saccharified solution having a glucose concentration of about 33.6 g / l was obtained.

[Comparative Example 2]
Suspended in a hydrolysis reaction vessel containing 20 mM citrate buffer pH 5.0 so that the mecanomi chemical-treated eucalyptus wood flour was 20% (w / v) and heat-treated at 121 ° C. for 30 minutes. After cooling to room temperature, add Cellulase T “Amano” 4 (Amano Enzyme) 1.8FPU / (g-substrate), Optimash BG (Genencor) 20μl / (g-substrate), 37 ° C, 120rpm For 120 hours. The hydrolyzate collected every 24 hours immediately after the start of the hydrolysis reaction was boiled for 3 minutes to completely deactivate the enzyme, and centrifuged at 15000 rpm for 10 minutes to recover the water-soluble part. The recovered water-soluble part was then analyzed by high performance liquid chromatography (HPLC) to determine the glucose concentration. FIG. 4 shows the glucose concentration in the hydrolysis test of Comparative Example 2. From the figure, a saccharified solution having a glucose concentration of about 23.3 g / l was obtained.

[Example 3]
Murakia brolopis (FERM P-22126 strain) 8 which had been pre-cultured in YPD liquid medium (10 g / l yeast extract, 20 g / l peptone, 20 g / l glucose) for 96 hours at 10 ° C. and 120 rpm The ml was centrifuged at 3000 × g 4 ° C. for 10 minutes to recover the cells. The bacterial cells collected after washing twice with sterilized distilled water and centrifuging again were suspended in 1 ml of sterilized distilled water, and prepared as an inoculation source for Murakia brolopis.

100 g / l, 5 g / l yeast extract, 5 g / l Bacto peptone, 2 g / l NH ₄ Cl, 1 g / l mechanochemically treated eucalyptus wood flour in 20 mM citrate buffer (pH 5.0) KH ₂ PO ₄ , 0.3 g / l MgSO ₄・ 7H ₂ O, Nonionic surfactant Tween 80 to 1% (v / v) of simultaneous saccharification and fermentation reaction solution, 5 U / g substrate Add the lipolytic enzyme lipase AS “Amano”, inoculate the inoculation source of Murakia brolopis as described above, cellulase T “Amano” 4 (Amano Enzyme) 9 FPU / g, Genencor Optimash BG 20 μl / g was added, and simultaneous saccharification reaction was performed with stirring at 10 ° C. and 180 rpm for 120 hours. A part of the simultaneous saccharification and fermentation test solution was collected immediately after the start of the simultaneous saccharification and fermentation test, centrifuged at 12100 × g at 4 ° C. for 5 minutes, and the supernatant was collected.

The collected supernatant was then measured for glucose concentration and ethanol concentration by high-speed chromatography. The quantitative conditions for glucose were as follows: detector: JASCO RI-2031, column: Aminex HPX87P, mobile phase: ion-exchanged water, flow rate: 0.6 mL / min, column temperature: 80 ° C. The ethanol quantification conditions were as follows: detector: JASCO RI-2031, column: Aminex HPX87H, mobile phase: 5 mM H ₂ SO ₄ , flow rate: 0.6 mL / min, column temperature: 65 ° C. The results are shown in FIG. About 10.3 g / l of glucose was produced 24 hours after the start of the simultaneous saccharification and fermentation test, and thereafter the glucose concentration remained almost constant. Finally, 2.8 g / l glucose remained, and a maximum of 17.3 g / l ethanol could be obtained.

[Comparative Example 3]
200 g / l, 5 g / l yeast extract, 5 g / l Bacto peptone, 2 g / l NH ₄ Cl, 1 g / l mechanochemically treated eucalyptus wood flour in 20 mM citrate buffer (pH 5.0) After inoculating KH ₂ PO ₄ , 0.3 g / l MgSO ₄ .7H ₂ O and inoculating the inoculum cultured under the conditions of Example 3, cellulase T “Amano” 4 (Amano Enzyme Co., Ltd.) 9 FPU / (g-substrate), Genencor Optimash BG 20 μl / (g-substrate) was added, and simultaneous saccharification reaction was performed with stirring at 10 ° C. and 180 rpm for 120 hours. A part of the simultaneous saccharification and fermentation test solution was collected immediately after the start of the simultaneous saccharification and fermentation test, centrifuged at 12100 × g at 4 ° C. for 5 minutes, and the supernatant was collected.

  The collected supernatant was then subjected to measurement of glucose concentration and ethanol concentration by high-speed chromatography under the conditions of Example 1. The results are shown in FIG. About 11.9 g / l of glucose was produced 24 hours after the start of the simultaneous saccharification and fermentation test, and thereafter the glucose concentration remained almost constant. Finally, 14.2 g / l glucose remained, and a maximum of 7.1 g / l ethanol could be obtained.

  According to the present invention, for example, a lipid used for an enzymatic saccharification reaction by adding a lipolytic enzyme or a nonionic surfactant and a lipolytic enzyme to a hydrolysis vessel containing mechanochemically treated lignocellulosic biomass. The amount of degrading enzyme can be reduced to 1/5 or less. Finally, about twice as much glucose as the conventional enzyme saccharification reaction without adding nonionic surfactant or lipolytic enzyme Can be obtained. Moreover, by adding a nonionic surfactant and a lipolytic enzyme to the reaction vessel for simultaneous saccharification and fermentation, the ethanol production can be increased by about 2.5 times compared to the conventional simultaneous saccharification and fermentation. Not only can the saccharification reaction cost and simultaneous saccharification fermentation cost of lignocellulosic biomass be reduced, but the obtained sugar can be used as a fermentation raw material used for lactic acid, bioethanol, biodiesel production and the like.

Claims (3)

  In the method for producing sugar from lignocellulosic biomass using an enzyme, the yield of sugar is improved by adding a lipolytic enzyme to the enzymatic saccharification step.   The method according to claim 1, wherein a nonionic surfactant is also added in addition to the lipolytic enzyme.   In the method for producing ethanol from lignocellulosic biomass by simultaneous saccharification and fermentation, the method improves the ethanol yield by adding a lipolytic enzyme or a lipolytic enzyme and a nonionic surfactant to the simultaneous saccharification and fermentation step.

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