JP5681923B2 - Method for producing high-concentration saccharified solution - Google Patents
Method for producing high-concentration saccharified solution Download PDFInfo
- Publication number
- JP5681923B2 JP5681923B2 JP2010075425A JP2010075425A JP5681923B2 JP 5681923 B2 JP5681923 B2 JP 5681923B2 JP 2010075425 A JP2010075425 A JP 2010075425A JP 2010075425 A JP2010075425 A JP 2010075425A JP 5681923 B2 JP5681923 B2 JP 5681923B2
- Authority
- JP
- Japan
- Prior art keywords
- biomass
- reaction
- solvent
- water
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000004519 manufacturing process Methods 0.000 title claims description 13
- 239000002028 Biomass Substances 0.000 claims description 47
- 235000000346 sugar Nutrition 0.000 claims description 40
- 239000003960 organic solvent Substances 0.000 claims description 36
- 239000007810 chemical reaction solvent Substances 0.000 claims description 30
- 102000004190 Enzymes Human genes 0.000 claims description 25
- 108090000790 Enzymes Proteins 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 239000003125 aqueous solvent Substances 0.000 claims description 22
- 238000000605 extraction Methods 0.000 claims description 13
- 230000002209 hydrophobic effect Effects 0.000 claims description 11
- 150000001720 carbohydrates Chemical class 0.000 claims description 9
- 238000000354 decomposition reaction Methods 0.000 claims description 6
- 150000004676 glycans Chemical class 0.000 claims description 6
- 229920001282 polysaccharide Polymers 0.000 claims description 6
- 239000005017 polysaccharide Substances 0.000 claims description 6
- 238000011084 recovery Methods 0.000 claims description 5
- 150000008163 sugars Chemical class 0.000 claims description 4
- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 96
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 55
- 238000012360 testing method Methods 0.000 description 28
- 239000007853 buffer solution Substances 0.000 description 27
- 239000000872 buffer Substances 0.000 description 24
- 229940088598 enzyme Drugs 0.000 description 22
- 239000000243 solution Substances 0.000 description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 21
- 238000000034 method Methods 0.000 description 19
- 241000218645 Cedrus Species 0.000 description 13
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 13
- 239000008103 glucose Substances 0.000 description 13
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 12
- 239000011259 mixed solution Substances 0.000 description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 12
- 229920002678 cellulose Polymers 0.000 description 11
- 239000001913 cellulose Substances 0.000 description 11
- 239000000203 mixture Substances 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 230000002255 enzymatic effect Effects 0.000 description 9
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 7
- 239000012071 phase Substances 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 241000227653 Lycopersicon Species 0.000 description 5
- 150000002772 monosaccharides Chemical class 0.000 description 5
- 239000007788 liquid Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 108010059892 Cellulase Proteins 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 3
- KXKVLQRXCPHEJC-UHFFFAOYSA-N acetic acid trimethyl ester Natural products COC(C)=O KXKVLQRXCPHEJC-UHFFFAOYSA-N 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 229940106157 cellulase Drugs 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 238000004821 distillation Methods 0.000 description 3
- 239000000446 fuel Substances 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- 239000004382 Amylase Substances 0.000 description 2
- 102000013142 Amylases Human genes 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 2
- 108010008885 Cellulose 1,4-beta-Cellobiosidase Proteins 0.000 description 2
- RGSFGYAAUTVSQA-UHFFFAOYSA-N Cyclopentane Chemical compound C1CCCC1 RGSFGYAAUTVSQA-UHFFFAOYSA-N 0.000 description 2
- 229920002488 Hemicellulose Polymers 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- 240000003768 Solanum lycopersicum Species 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 235000019418 amylase Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 108010002430 hemicellulase Proteins 0.000 description 2
- 229940059442 hemicellulase Drugs 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- QWTDNUCVQCZILF-UHFFFAOYSA-N isopentane Chemical compound CCC(C)C QWTDNUCVQCZILF-UHFFFAOYSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
- 229930195734 saturated hydrocarbon Natural products 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 1
- 240000005109 Cryptomeria japonica Species 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- 101710156496 Endoglucanase A Proteins 0.000 description 1
- 101710112457 Exoglucanase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 102100026367 Pancreatic alpha-amylase Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 108090000637 alpha-Amylases Proteins 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 108010019077 beta-Amylase Proteins 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 108010091371 endoglucanase 1 Proteins 0.000 description 1
- 108010091384 endoglucanase 2 Proteins 0.000 description 1
- 108010092450 endoglucanase Z Proteins 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 230000009229 glucose formation Effects 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 150000008282 halocarbons Chemical class 0.000 description 1
- DMEGYFMYUHOHGS-UHFFFAOYSA-N heptamethylene Natural products C1CCCCCC1 DMEGYFMYUHOHGS-UHFFFAOYSA-N 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 235000011837 pasties Nutrition 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Processing Of Solid Wastes (AREA)
Description
本発明は、バイオマスから糖化液を製造する方法に関する。更に詳しくは、疎水性の有機溶媒を用いて、バイオマスを用いた燃料やマテリアルの製造・開発等に適した、高濃度糖化液を低コストで容易に製造する方法に関する。 The present invention relates to a method for producing a saccharified solution from biomass. More specifically, the present invention relates to a method for easily producing a high-concentration saccharified solution at low cost, which is suitable for production and development of fuels and materials using biomass using a hydrophobic organic solvent.
脱石油化学社会の構築には、バイオマスと称される生物由来の資源を活用したエネルギーやマテリアルの生産は不可欠である。中でも太陽エネルギーと二酸化炭素を光合成により有効活用できる植物系のバイオマス（以下、バイオマスという）は、大気中の二酸化炭素濃度増加を招くことのない、所謂カーボンニュートラルな資源として有効活用が望まれている。 In order to build a de-petrochemical society, it is essential to produce energy and materials using biological resources called biomass. Among them, plant-based biomass (hereinafter referred to as biomass) that can effectively utilize solar energy and carbon dioxide by photosynthesis is desired to be effectively utilized as a so-called carbon-neutral resource that does not increase the concentration of carbon dioxide in the atmosphere. .
一方で、これらバイオマス由来のマテリアルやエネルギーのコスト高が、市場普及への大きな障害となっている。バイオマス由来の製品を製造する場合、多額のコストを要する多くの工程を経る。例えばセルロース系バイオマスからエタノール等のエネルギーを生産する場合、次の工程を経る。
まず、１．木材や草などの原材料を粉砕・加圧・加熱処理等を行った後、セルロースやヘミセルロースと言った多糖類を取り出す。次に、２．多糖類を酸や酵素などの触媒により加水分解すなわち糖化して、グルコースなどの単糖類を回収する。３．得られた単糖類は、酵母などの微生物の発酵によりエタノールに変換する。さらに、４．エタノールと共存する多量の水を除去するために、蒸留や脱水を行う。
また、ポリ乳酸などのマテリアルをバイオマスから生産する場合も、同様の工程を経て、得られたグルコースなどの単糖類を用いる。
On the other hand, the high cost of these biomass-derived materials and energy is a major obstacle to market penetration. When manufacturing a product derived from biomass, it goes through many processes that require a large amount of cost. For example, when energy such as ethanol is produced from cellulosic biomass, the following steps are performed.
First, 1. After pulverizing, pressing, and heat-treating raw materials such as wood and grass, polysaccharides such as cellulose and hemicellulose are taken out. Next, 2. The polysaccharide is hydrolyzed, that is, saccharified with a catalyst such as an acid or an enzyme to recover a monosaccharide such as glucose. 3. The obtained monosaccharide is converted into ethanol by fermentation of microorganisms such as yeast. Furthermore, 4. Distillation and dehydration are performed to remove a large amount of water that coexists with ethanol.
In addition, when a material such as polylactic acid is produced from biomass, a monosaccharide such as glucose obtained through the same process is used.
粉砕後のバイオマスを上記２の酵素加水分解反応すなわち酵素糖化反応する場合、試料と酵素の接触回数を向上させるために、水（溶媒）を加えて分散させる。例えばセルロース系バイオマス試料の場合、試料の吸水性が良く少量の液体では分散しにくいため、少なくとも試料の１０〜２０倍の水を加える必要がある（試料濃度：５〜１０ｗ／ｖ％程度）。この様に多くの水を加えるため、加水分解（糖化）によって得られる糖液の濃度は低くなり、３の発酵時には糖の１/３が二酸化炭素になり減少するために、エタノール濃度は３〜７％程度と低濃度になる。 When the pulverized biomass is subjected to the enzyme hydrolysis reaction (2), that is, the enzyme saccharification reaction, water (solvent) is added and dispersed in order to improve the number of contact between the sample and the enzyme. For example, in the case of a cellulosic biomass sample, it is necessary to add at least 10 to 20 times as much water as the sample (sample concentration: about 5 to 10 w / v%) because the sample absorbs well and is difficult to disperse with a small amount of liquid. Since a lot of water is added in this way, the concentration of the sugar solution obtained by hydrolysis (saccharification) is low, and 1/3 of the sugar is reduced to carbon dioxide during the fermentation of 3, so that the ethanol concentration is 3 to 3. As low as 7%.
バイオ燃料としてエタノールを用いる場合、得られた低濃度のエタノールを４の蒸留・脱水を行いエタノールの濃度を９０％以上にまで濃縮することになる。これまでの方法では、この工程で消費される熱エネルギーが非常に大きくなるばかりでなく、糖化の後工程となる３及び４の装置が不要な水を入れるために大型化し、製品のコスト高を招いている。このことは、バイオマテリアルを製造する場合も、グルコースから乳酸・コハク酸などへ発酵させることから、エタノールの場合と同様の問題を起こしている。
斯かる状況に鑑み、本発明は工業的に安価かつ容易にバイオマスを酵素により糖化し、共存する水割合が少ない高濃度の糖化液を取得する方法を提供することを課題とする。
When ethanol is used as the biofuel, the obtained low-concentration ethanol is distilled and dehydrated 4 to concentrate the ethanol concentration to 90% or more. In the conventional methods, not only the heat energy consumed in this process is very large, but also the devices 3 and 4 that are the post-saccharification process are increased in size to contain unnecessary water, which increases the cost of the product. Invited. This also causes the same problem as in the case of ethanol since biomaterials are fermented from glucose to lactic acid and succinic acid.
In view of such a situation, an object of the present invention is to provide a method for obtaining a high-concentration saccharified solution with a small proportion of water present by saccharifying biomass with an enzyme and industrially inexpensively and easily.
本発明者は、上記課題を解決するために種々研究を重ねた結果、バイオマスの酵素加水分解（糖化）において、反応溶媒中に疎水性の有機溶媒を存在させることにより、高効率に分解（糖化）が起こることを見出し、本発明に至った。従って、本発明は、以上の知見に基づいたものであり、次の構成からなる。 The present inventor has conducted various studies to solve the above-mentioned problems, and as a result, in the enzymatic hydrolysis (saccharification) of biomass, the presence of a hydrophobic organic solvent in the reaction solvent enables high-efficiency degradation (saccharification). ) Occurred, and the present invention was achieved. Therefore, the present invention is based on the above knowledge and has the following configuration.
［１］バイオマスから酵素を用いて糖化液を製造する方法であって、
疎水性の有機溶媒が存在する反応溶媒中に、バイオマス及び酵素を添加して攪拌することにより、前記バイオマス中の多糖類をより低分子の糖類に加水分解する分解ステップと、
必要により前記分解ステップの最終段階で前記反応溶媒中に水系溶媒を添加して、前記分解されたより低分子の糖類を水系溶媒に抽出する抽出ステップと、
前記水系溶媒に抽出された糖類を糖化液として回収する回収ステップと、
を含むことを特徴とする糖化液を製造する製造方法である。
[1] A method for producing a saccharified solution from biomass using an enzyme,
In a reaction solvent in which a hydrophobic organic solvent is present, a biomass and an enzyme are added and stirred to hydrolyze the polysaccharide in the biomass into lower molecular weight saccharides;
An extraction step in which an aqueous solvent is added to the reaction solvent in the final stage of the decomposition step as necessary, and the decomposed lower molecular sugars are extracted into the aqueous solvent;
A recovery step of recovering the saccharide extracted in the aqueous solvent as a saccharified solution;
Is a production method for producing a saccharified solution.
本発明における「バイオマス」とは、植物系のバイオマスを意味し、セルロース系バイオマス、澱粉系バイオマス等を含むが、多糖類を含むバイオマスであればこれらに限定されない。本発明における「多糖類」とは、セルロース、ヘミセルロース等を含むが、これらに限定されない。 “Biomass” in the present invention means plant-based biomass and includes cellulosic biomass, starch-based biomass, and the like, but is not limited thereto as long as it includes polysaccharides. The “polysaccharide” in the present invention includes, but is not limited to, cellulose, hemicellulose and the like.
［２］前記分解ステップにおいて、前記反応溶媒は前記有機溶媒と水系溶媒との混合液であり、該混合液中に前記バイオマスは分散されている、ことを特徴とする［１］に記載の方法である。
本発明における「水系溶媒」とは、水のみ、及び水を含む緩衝液等を含むが、水を含んでいればよい。
[2] The method according to [1], wherein, in the decomposition step, the reaction solvent is a mixed solution of the organic solvent and an aqueous solvent, and the biomass is dispersed in the mixed solution. It is.
The “aqueous solvent” in the present invention includes only water and a buffer solution containing water, but may contain water.
本発明における「分散されている」とは、液相の反応溶媒へバイオマスの粉体を混入させた状態を指し、この状態でバイオマスの粉体は反応溶媒中で自由運動が可能である（図１ｂ参照）。
有機溶媒の種類、バイオマスの種類、バイオマスの粒径、有機溶媒と水系溶媒との配合比、反応溶媒とバイオマスとの混合比等の要因によってはバイオマスと反応溶媒との混合物がペースト状（高粘度流体）となる場合があり、このペースト状態ではバイオマスに対する酵素の接触頻度が低下して充分な加水分解速度を得難い（図１ａ）。
従って、これらの要因を適宜調整して、バイオマスを反応溶媒において分散させ、反応溶媒中の酵素がバイオマスに対して自由に接触できるようにする。
“Dispersed” in the present invention refers to a state in which biomass powder is mixed in a liquid phase reaction solvent, and in this state, the biomass powder can freely move in the reaction solvent (FIG. 1b).
Depending on factors such as the type of organic solvent, the type of biomass, the particle size of the biomass, the mixing ratio of the organic solvent and the aqueous solvent, and the mixing ratio of the reaction solvent and the biomass, the mixture of the biomass and the reaction solvent is pasty (high viscosity In this paste state, the contact frequency of the enzyme to the biomass is lowered, and it is difficult to obtain a sufficient hydrolysis rate (FIG. 1a).
Therefore, these factors are appropriately adjusted to disperse the biomass in the reaction solvent so that the enzyme in the reaction solvent can freely contact the biomass.
バイオマスを分散状態とするには上記要因を適宜調整するものであるが、本発明者の検討によれば、反応溶媒としてその水系溶媒の配合量を３５容量％以下としたもの、を採用することが好ましい（本発明の第[３]局面）。
ここに、「前記水系溶媒は３５容量％以下」とは、前記水系溶媒を含まない場合を含む。
この範囲を超えて水系溶媒を加えることも勿論可能であるが、アルコール回収時の蒸留量を考慮すると３５容量％以下が適当と考えられる。なお、この範囲を大きく超えて水系溶媒を配合量すると、バイオマスと反応溶媒との混合物はペースト状になる可能性が高い。
In order to make the biomass in a dispersed state, the above factors are appropriately adjusted. According to the study of the present inventor, a reaction solvent having an aqueous solvent content of 35% by volume or less should be adopted. Is preferable (the [3] aspect of the present invention).
Here, “the aqueous solvent is 35% by volume or less” includes a case where the aqueous solvent is not included.
Of course, it is possible to add the aqueous solvent beyond this range, but considering the amount of distillation at the time of alcohol recovery, 35% by volume or less is considered appropriate. In addition, when the amount of the aqueous solvent exceeds the range, the mixture of the biomass and the reaction solvent is likely to become a paste.
［４］前記分解ステップにおいて、反応開始時は前記反応溶媒として前記有機溶媒を用い、反応の途中で水系溶媒を添加する、ことを特徴とする［１］に記載の方法である。
［５］前記酵素は固定化酵素であることを特徴とする［１］〜［４］のいずれかに記載の方法である。
従来、バイオマスの糖化反応には加水するための水を分散溶媒として用いたが、本発明による方法では試料の水分、もしくは反応に最低限必要とされる水分を分散用の疎水性の有機溶媒に投入することにより行うことができる。酵素糖化反応時の水分量が糖類の抽出・回収に十分な量含まれている場合、抽出・回収時に水系溶媒の添加は必要でなくなる。なお、投入する水系溶媒の量は抽出用も含め、最大で前記反応溶媒の３５容量％までが好ましく、１容量％以上〜３０容量％以下がより好ましく、１５容量％以上〜２５容量％以下では、水系溶媒単独よりも酵素活性が顕著に高められるので、特に好ましい。水系溶媒―前記有機溶媒（前記反応溶媒）の１００容量部に対して試料約５〜１０重量部となることが好ましい。
[4] The method according to [1], wherein in the decomposition step, the organic solvent is used as the reaction solvent at the start of the reaction, and an aqueous solvent is added during the reaction.
[5] The method according to any one of [1] to [4], wherein the enzyme is an immobilized enzyme.
Conventionally, water used for saccharification of biomass has been used as a dispersion solvent. However, in the method according to the present invention, the moisture of the sample or the minimum water required for the reaction is used as a hydrophobic organic solvent for dispersion. It can be done by throwing in. When the amount of water during the enzymatic saccharification reaction is sufficient for extraction / recovery of saccharides, it is not necessary to add an aqueous solvent during extraction / recovery. The amount of the aqueous solvent to be added, including for extraction, is preferably up to 35% by volume of the reaction solvent, more preferably 1% by volume to 30% by volume, and more preferably 15% by volume to 25% by volume. Since the enzyme activity is remarkably enhanced as compared with the aqueous solvent alone, it is particularly preferable. It is preferable that the sample is about 5 to 10 parts by weight with respect to 100 parts by volume of the aqueous solvent-the organic solvent (the reaction solvent).
本発明に用いる疎水性の有機溶媒は、水と相溶性の無いものであればいずれも使用可能であるが、具体的には、
鎖状飽和炭化水素系溶媒： ｎ−ペンタン、２−メチルブタン、ｎ−ヘキサン、へプタン、オクタンなど
環状飽和炭化水素系溶媒： シクロペンタン、シクロヘキサンなど
芳香族系炭化水素系溶媒： ベンゼン、トルエン、キシレン、エチルベンゼンなど
その他（ハロゲン化炭化水素系溶剤、エーテル、エステル類、石油留分など） ： クロロホルム、四塩化炭素、酢酸エチル、酢酸メチルなど
が用いられるが、酢酸メチルやトルエンなど水と完全に２層分離する溶媒が望ましい。
Any hydrophobic organic solvent used in the present invention can be used as long as it is not compatible with water. Specifically,
Chain saturated hydrocarbon solvents: n-pentane, 2-methylbutane, n-hexane, heptane, octane and other cyclic saturated hydrocarbon solvents: cyclopentane, cyclohexane and other aromatic hydrocarbon solvents: benzene, toluene, xylene Others (halogenated hydrocarbon solvents, ethers, esters, petroleum fractions, etc.): Chloroform, carbon tetrachloride, ethyl acetate, methyl acetate, etc. are used, but water such as methyl acetate and toluene is completely 2 Solvents that separate layers are desirable.
本発明に用いる酵素（加水分解酵素）としては、セルロース系バイオマスの場合、セルラーゼ、ヘミセルラーゼが好適なものとして挙げられ、澱粉系バイオマスの場合アミラーゼが好適なものとして挙げられるが、これに縛られるものではない。
中でも、セルラーゼとしては、エンドグルカナーゼ ＥＣ３．２．１．４、エキソグルカナーゼ（セロビオヒドロラーゼ） ＥＣ ３．２．１．９１及びβ-グルコシダーゼＥＣ３．２．１.２１の混合が望ましい。アミラーゼとしては、α-アミラーゼ ＥＣ ３．２．１．１、β-アミラーゼＥＣ ３．２．１．２、グルコアミラーゼＥＣ ３．２．１．３の混合が望ましい。
As the enzyme (hydrolyzing enzyme) used in the present invention, cellulase and hemicellulase are preferable for cellulosic biomass, and amylase is preferable for starch biomass. It is not a thing.
Among them, the cellulase is preferably a mixture of endoglucanase EC 3.2.1.4, exoglucanase (cellobiohydrolase) EC 3.2.1.91 and β-glucosidase EC 3.2.1.21. As the amylase, α-amylase EC 3.2.1.1, β-amylase EC 3.2.1.2, and glucoamylase EC 3.2.1.3 are desirable.
これら酵素は、疎水性の有機溶媒による失活の影響を抑える目的から、不溶性の担体に固定化して固定化酵素として用いることが望ましい。酵素の固定化方法は、イオン結合法、物理吸着法、化学結合法、架橋法及び包括法があるが、本発明では担体の耐久性が高く使用中に酵素が担体外に放出されにくい、シリカゲルの包括固定化剤が望ましい。 These enzymes are preferably immobilized on an insoluble carrier and used as an immobilized enzyme for the purpose of suppressing the influence of inactivation by a hydrophobic organic solvent. Enzyme immobilization methods include ionic bond method, physical adsorption method, chemical bond method, cross-linking method and inclusion method. In the present invention, the carrier has high durability, and the enzyme is not easily released from the carrier during use. The entrapping immobilization agent is desirable.
本発明の反応温度は４０〜５０℃が最適であるが、これに限るものではなく、酵素の至適温度や耐久性により適切に設定することが可能である。抽出及び反応時に使用する水系溶媒は、ｐＨ５〜７の緩衝液が望ましいが、これに拘るものではない。本発明では、例えば疎水性の有機溶媒中にバイオマス、酵素を投入し攪拌しながら分散・反応させ、バイオマスから低分子の糖類を得る。その後に最低量の緩衝液を投入し攪拌しながら、緩衝液に低分子の糖類を抽出させ高濃度の糖溶液を取得する。反応後、攪拌を停止させると前記有機溶媒と水は比重差により２層に分離し、目的とする低分子の糖化液が容易に回収できる。回収した糖化液のうち単糖などの糖液は、糖液は発酵槽でエタノールに変換される。さらに、蒸留・濃縮行程を経て９０％以上のエタノール燃料となる。 The reaction temperature of the present invention is optimally 40 to 50 ° C., but is not limited thereto, and can be appropriately set depending on the optimum temperature and durability of the enzyme. The aqueous solvent used at the time of extraction and reaction is preferably a buffer solution having a pH of 5 to 7, but is not limited thereto. In the present invention, for example, biomass and an enzyme are introduced into a hydrophobic organic solvent and dispersed and reacted while stirring to obtain a low-molecular sugar from the biomass. Thereafter, a minimum amount of a buffer solution is added, and while stirring, a low-molecular sugar is extracted from the buffer solution to obtain a high-concentration sugar solution. When the stirring is stopped after the reaction, the organic solvent and water are separated into two layers due to the difference in specific gravity, and the intended low-molecular saccharified solution can be easily recovered. Among the collected saccharified liquid, sugar liquid such as monosaccharide is converted into ethanol in the fermenter. Furthermore, it becomes 90% or more ethanol fuel through the distillation and concentration process.
本発明によれば、従来の糖化液の製造手法に比べて、高濃度糖化液を、低コストで容易に製造する方法が提供される。これにより、バイオマス由来のマテリアルやエネルギーにおけるコスト高や煩雑さ等の問題を解決することができ、バイオマスを用いた燃料やマテリアルの製造・開発分野において極めて有意義である。 According to the present invention, a method for easily producing a high-concentration saccharified solution at a low cost as compared with a conventional method for producing a saccharified solution is provided. Thereby, problems such as high cost and complexity in biomass-derived materials and energy can be solved, and this is extremely significant in the field of production and development of fuels and materials using biomass.
（Ａ）試験方法
１．試料及び試薬
セルロース系バイオマス試料には結晶性セルロースのセオラスTG（旭化成（株））、トマトの葉茎の廃材及びスギを用いた。なお、トマトの葉茎及びスギは、乾式粉砕後に篩い分けを行い、45μmから90μmの分画サイズを用いた。セルロース糖化用酵素には、セルラーゼA「アマノ」３、セルラーゼＴ「アマノ」４、及びヘミセルラーゼ「アマノ」90 （全て、天野エンザイム（株）製）を同量混合して用いた。
(A) Test method Samples and Reagents Cellulose biomass samples used were the crystalline cellulose Theolas TG (Asahi Kasei Co., Ltd.), tomato leaf stem waste and cedar. Tomato leaves and cedar were sieved after dry pulverization, and a fraction size of 45 μm to 90 μm was used. Cellulase A “Amano” 3, cellulase T “Amano” 4 and hemicellulase “Amano” 90 (all from Amano Enzyme Co., Ltd.) were mixed and used as the enzyme for cellulose saccharification.
２．試料の水分測定
試料の水分測定は105℃の乾燥機を用いて行った。105℃で恒量になるまで乾燥した試料の重量を用い、以下の式により試料の水分を算出した。
試料の水分（重量％）＝（乾燥前の試料の重量(g) ― 105℃で乾燥した試料の重量(g)）／（乾燥前の試料の重量(g)）×100％
2. Water content measurement of sample The water content of the sample was measured using a dryer at 105 ° C. Using the weight of the sample dried to a constant weight at 105 ° C., the moisture of the sample was calculated by the following formula.
Sample moisture (% by weight) = (weight of sample before drying (g) −weight of sample dried at 105 ° C. (g)) / (weight of sample before drying (g)) × 100%
（Ｂ）酵素糖化反応
疎水性の有機溶媒の入ったガラス製容器に試料及び酵素を投入し、50℃の環境下において180rpmで3日間振とう・反応させた。なお、酵素量は試料1gあたり各20mg、前記有機溶媒は試料0.5gあたり10mLとした。反応時に水を投入する場合は、pH 5.0のMacIlvain緩衝液を用い、投入した前記緩衝液と同量の有機溶媒を除いて、溶液量を10mL に統一して試験に供した。
(B) Enzymatic saccharification reaction A sample and an enzyme were placed in a glass container containing a hydrophobic organic solvent, and the mixture was shaken and reacted at 180 rpm in a 50 ° C environment for 3 days. The enzyme amount was 20 mg per 1 g of the sample, and the organic solvent was 10 mL per 0.5 g of the sample. When water was added during the reaction, a MacIlvain buffer solution having a pH of 5.0 was used, and the same amount of the organic solvent as the buffer solution was removed, and the solution volume was unified to 10 mL and used for the test.
（Ｃ）糖類の抽出
反応の最終段階でガラス容器にイオン交換水を投入し、密栓後1分間強く攪拌し低分子の糖類を水に抽出させた。抽出に使用した水の量は試料0.5gに対し2ｍLとした。なお、反応時に水を投入した系では、抽出用の水量を減らし、反応時の水と抽出用の水が併せて試料0.5gに対し2mL となるように統一した。
（Ｄ）成分分析
グルコースの定量分析は、グルコースＣIIテストワコー（和光純薬（株）製）を用いて行った。全糖の分析はフェノール硫酸法を用いた。
(C) Extraction of saccharides In the final stage of the reaction, ion-exchanged water was put into a glass container, and after tight sealing, the mixture was vigorously stirred for 1 minute to extract low-molecular-weight saccharides into water. The amount of water used for extraction was 2 mL per 0.5 g sample. In the system in which water was added during the reaction, the amount of water used for extraction was reduced, and the water used during the reaction and the water used for extraction were combined so that the total volume was 2 mL per 0.5 g sample.
(D) Component analysis Quantitative analysis of glucose was performed using Glucose CII Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The phenol-sulfuric acid method was used for the analysis of total sugar.
（Ｅ）実験結果
１−１．スギ混濁液の粘度
密閉容器中のスギにMacIlvain緩衝液又はトルエンを投入したところ、前者は後者に比べ粘度が大きく上昇した。スギ2gに対し前記緩衝液9mLを加えた状態を図１ａに示し、同じくトルエン9mLを加えた状態を図１ｂに示す。各々の粘度をB型粘度計で測定したところ、前記緩衝液を用いた試験区では2,400から3,000cP、トルエンを用いた試験区では625から750cPであった。
(E) Experimental results 1-1. Viscosity of cedar turbid liquid When MacIlvain buffer or toluene was added to cedar in a sealed container, the viscosity of the former increased significantly compared to the latter. A state where 9 mL of the buffer solution is added to 2 g of cedar is shown in FIG. 1 a, and a state where 9 mL of toluene is also added is shown in FIG. Each viscosity was measured with a B-type viscometer and found to be 2,400 to 3,000 cP in the test group using the buffer solution and from 625 to 750 cP in the test group using toluene.
前記粘度及び図１から明らかなように、反応溶媒が同量であるにもかかわらず、前記緩衝液を用いた試験区では試料が前記容器を傾けても移動しないほど膨潤するのに対して（ペースト状態：図１ａ）、トルエンを用いた試験区では反応溶媒が2層に分離し試料が前記容器を傾けると試料粒子が移動する程度に分散された（分散状態：図１ｂ）。 As is apparent from the viscosity and FIG. 1, in the test section using the buffer solution, the sample swells so that it does not move even when the container is tilted despite the same amount of the reaction solvent ( Paste state: FIG. 1a), in the test section using toluene, the reaction solvent was separated into two layers, and the sample was dispersed to such an extent that the sample particles moved when the container was tilted (dispersion state: FIG. 1b).
１−２．各種有機溶媒中でのスギの糖化試験結果
pH 5.0のMacIlvain緩衝液、酢酸メチル、ｎ−ヘキサン、クロロホルム、及びトルエン中でスギ（0.5ｇ）を酵素糖化反応させた。また、容積比で緩衝液：有機溶媒＝1:9の溶液中でも同様の試験を行った。用いたスギの水分は12.1重量％であった。3日間の酵素糖化により生成した全糖のうち、抽出した水及び未抽出で使用した有機溶媒中に残った糖の割合、すなわち、各種有機溶媒中で糖化した後の水抽出全糖及び有機溶媒残存全糖の割合を表1に示す。全ての系において、90%以上の糖が水に抽出された。反応溶媒に緩衝液：有機溶媒＝1:9の混合液を用いた場合、水中に回収される糖の量が多くなった。
1-2. Results of saccharification test of cedar in various organic solvents
Sugi (0.5 g) was subjected to an enzymatic saccharification reaction in MacIlvain buffer at pH 5.0, methyl acetate, n-hexane, chloroform, and toluene. The same test was performed even in a solution of buffer solution: organic solvent = 1: 9 by volume ratio. The water content of the cedar used was 12.1% by weight. Of the total sugars produced by enzymatic saccharification for 3 days, the proportion of sugar remaining in the extracted water and the organic solvent used without extraction, that is, the water-extracted total sugar and organic solvent after saccharification in various organic solvents The percentage of total remaining sugar is shown in Table 1. In all systems, over 90% of the sugar was extracted into water. When a mixed solution of buffer solution: organic solvent = 1: 9 was used as a reaction solvent, the amount of sugar recovered in water increased.
また、3日間反応後に抽出した水の全糖濃度を図２に示す。図２中、
１ pH 5.0のMacIlvain緩衝液のみ、
２ 酢酸エチルのみ、
３ ｎ−ヘキサンのみ、
４ クロロホルムのみ、
５ トルエンのみ、
６ 前記緩衝液：酢酸エチル＝１：９（容積比）の混合液、
７ 前記緩衝液：ｎ−ヘキサン＝１：９（容積比）の混合液、
８ 前記緩衝液：クロロホルム＝１：９（容積比）の混合液、
９ 前記緩衝液：トルエン＝１：９（容積比）の混合液である（以下、図３〜５も同様）。
Moreover, the total sugar concentration of the water extracted after reaction for 3 days is shown in FIG. In FIG.
1 MacIlvain buffer pH 5.0 only
2 Ethyl acetate only,
3 n-hexane only,
4 Chloroform only
5 Toluene only,
6 Mixed solution of the buffer: ethyl acetate = 1: 9 (volume ratio),
7 a mixed solution of the buffer solution: n-hexane = 1: 9 (volume ratio),
8 A mixture of the above buffer: chloroform = 1: 9 (volume ratio),
9 It is a mixed solution of the buffer solution: toluene = 1: 9 (volume ratio) (hereinafter, the same applies to FIGS. 3 to 5).
なお、有機溶媒を使用せずpH 5.0のMacIlvain緩衝液のみの試験区（１）は、反応後に水を加えることなく緩衝液の糖濃度を測定した。緩衝液中の糖化に比べ、有機溶媒中で糖化反応して水抽出した試験区は、全て高い濃度の糖液が回収された。中でも、水：有機溶媒＝1:9の溶液中で反応させた回収液の糖液濃度は高く、いずれの場合も、MacIlvain緩衝液のみの試験区（１）の3.0mg/mLに比べ約6倍の18mg/mL以上になった。 In addition, in the test section (1) containing only MacIlvain buffer having a pH of 5.0 without using an organic solvent, the sugar concentration of the buffer was measured without adding water after the reaction. Compared to the saccharification in the buffer solution, the saccharification reaction in the organic solvent and the water extracted from the test plots were all collected at a high concentration. In particular, the concentration of the sugar solution in the recovered solution reacted in a solution of water: organic solvent = 1: 9 is high, and in each case, it is about 6 compared to 3.0 mg / mL in the test section (1) with MacIlvain buffer only. Doubled 18 mg / mL or more.
図３に、上記試験の際に回収された全糖量を示す。MacIlvain緩衝液のみ中で酵素糖化した場合、30.2mgの糖が回収された。容積比で前記緩衝液：有機溶媒＝1:9の溶液中で試験を行った全ての場合において、酵素糖化で得られた糖の量が増加した。中でも、前記緩衝液：トルエン＝1:9の試験区（９）では、37.3mgと糖の量が最も多く、MacIlvain緩衝液中の1.1倍となった。 FIG. 3 shows the total amount of sugar collected during the test. When enzymatic saccharification was performed in MacIlvain buffer only, 30.2 mg of sugar was recovered. In all cases tested in a volume ratio of the above buffer: organic solvent = 1: 9 solution, the amount of sugar obtained by enzymatic saccharification increased. In particular, in the test group (9) where the buffer solution: toluene = 1: 9, the amount of sugar was 37.3 mg, which was 1.1 times that of the MacIlvain buffer solution.
２．各種有機溶媒中でのトマト葉茎の糖化試験結果
上記スギの試験と同様の試験方法で、トマトの葉茎（0.5ｇ）を酵素糖化した。使用したトマトの葉茎に含まれる水分は8.7重量％であった。3日間反応後に抽出した水の全糖濃度を図４に示す。なお、有機溶媒を使用せずpH 5.0のMacIlvain緩衝液のみの試験区（１）は、反応後に水を加えることなく緩衝液の糖濃度を測定した。スギの場合と同様に、前記緩衝液中の糖化に比べ、有機溶媒中で糖化反応して水で抽出した試験区は、全て高い濃度の糖液が回収された。中でも、水：有機溶媒＝1:9の溶液中で反応させた回収液の糖液濃度は高く、ｎ−ヘキサン、クロロホルム、及びトルエンを用いた場合、MacIlvain緩衝液のみの試験区（１）の3.４mg/mLに比べ約6倍の20mg/mL以上になった。
2. Results of saccharification test of tomato leaf stems in various organic solvents Tomato leaf stems (0.5 g) were enzymatically saccharified by the same test method as the cedar test described above. The water content of the tomato leaf stem used was 8.7% by weight. The total sugar concentration of the water extracted after the reaction for 3 days is shown in FIG. In addition, in the test section (1) containing only MacIlvain buffer having a pH of 5.0 without using an organic solvent, the sugar concentration of the buffer was measured without adding water after the reaction. As in the case of cedar, compared to the saccharification in the buffer solution, the saccharification reaction in an organic solvent and extraction with water all recovered high-concentration sugar solutions. Among them, the concentration of the sugar solution in the recovered solution reacted in a solution of water: organic solvent = 1: 9 is high. When n-hexane, chloroform, and toluene are used, the MacIlvain buffer only test group (1) Compared to 3.4 mg / mL, it was about 6 times higher than 20 mg / mL.
図５に、上記試験の際に回収された全糖量を示す。MacIlvain緩衝液のみ中で酵素糖化した場合、33.8mgの糖が回収された。容積比で緩衝液：有機溶媒＝1:9の溶液中で試験を行った全ての場合において、酵素糖化で得られた糖の量が増加した。中でも、ｎ−ヘキサン、クロロホルム、及びトルエンを用いた場合では、MacIlvain緩衝液の1.18倍を超える40mg以上の糖が得られた。 FIG. 5 shows the total amount of sugar recovered during the test. When enzymatic saccharification was performed in MacIlvain buffer only, 33.8 mg of sugar was recovered. In all cases tested in a volume ratio of buffer: organic solvent = 1: 9, the amount of sugar obtained by enzymatic saccharification increased. In particular, when n-hexane, chloroform, and toluene were used, 40 mg or more of saccharide exceeding 1.18 times that of MacIlvain buffer was obtained.
３．様々な水分量での結晶性セルロースの糖化試験結果
60℃の恒温器中でセオラスTGを58時間減圧乾燥し、水分値を4.11重量％とした。乾燥後のセオラスTG（0.5ｇ）をMacIlvain緩衝液とトルエンの比率を変えた様々な水分割合の溶液中で酵素糖化した。上記スギの試験と同様の試験方法で、3日間反応後に抽出した水中の全糖濃度を図６に、分解率を図７に示す。図６及び７中、
１ pH 5.0のMacIlvain緩衝液のみ、
２ トルエンのみ、
３ 前記緩衝液：トルエン＝０．４：９．６の混合液、
４ 前記緩衝液：トルエン＝０．６：９．４の混合液、
５ 前記緩衝液：トルエン＝０．８：９．２の混合液、
６ 前記緩衝液：トルエン＝１．０：９．０の混合液、
７ 前記緩衝液：トルエン＝１．５：８．５の混合液、
８ 前記緩衝液：トルエン＝２．０：８．０の混合液、
９ 前記緩衝液：トルエン＝２．２：７．８の混合液、
１０ 前記緩衝液：トルエン＝２．５：７．５の混合液、
１１ 前記緩衝液：トルエン＝３．０：７．０の混合液
であり、前記混合液は全て容積比で表す（以下、図８及び９も同様）。
3. Results of saccharification test of crystalline cellulose with various water contents
Theola TG was dried under reduced pressure for 58 hours in a 60 ° C. incubator, and the water content was adjusted to 4.11% by weight. The dried ceolas TG (0.5 g) was enzymatically saccharified in various water ratio solutions with different ratios of MacIlvain buffer and toluene. FIG. 6 shows the total sugar concentration in water extracted after the reaction for 3 days by the same test method as the above-mentioned cedar test, and FIG. 7 shows the degradation rate. 6 and 7,
1 MacIlvain buffer pH 5.0 only
2 Toluene only,
3 Buffer solution: toluene = 0.4: 9.6 mixed solution,
4 Mixture of the buffer: toluene = 0.6: 9.4,
5 Buffer solution: Toluene = 0.8: 9.2 mixed solution,
6 Buffer solution: Toluene = 1.0: 9.0 mixed solution,
7 Buffer solution: Toluene = 1.5: 8.5 mixture,
8 Buffer solution: Toluene = 2.0: 8.0 mixed solution,
9 Mixture of the buffer solution: toluene = 2.2: 7.8,
10 Buffer solution: Toluene = 2.5: 7.5 mixture,
11 Buffer solution: toluene = 3.0: 7.0 mixed solution, and all the mixed solutions are represented by volume ratio (hereinafter, the same applies to FIGS. 8 and 9).
有機溶媒を用いた試験区の全糖濃度は、MacIlvain緩衝液のみの試験区（１）より全て高くなった。特にMacIlvain緩衝液：トルエン＝1.5：8.5の試験区（７）では最大値30.1mgを示した。また、トルエンのみの場合（２）を除き、MacIlvain緩衝液を添加した試験区では、分解率が大きく向上した。 The total sugar concentration of the test group using the organic solvent was all higher than that of the test group (1) having only the MacIlvain buffer solution. In particular, the test group (7) with MacIlvain buffer: toluene = 1.5: 8.5 showed a maximum value of 30.1 mg. In addition, except for the case of toluene alone (2), the degradation rate was greatly improved in the test plots to which MacIlvain buffer was added.
さらに、同一の試験において全糖に含まれるグルコースについて分析を行った。グルコース濃度を図８に、グルコース量を元に算出したα- セルロース分解率を図９に示す。抽出水のグルコース濃度はトルエンのみで反応を行った系以外は全てMacIlvain緩衝液のみの試験区より高くなった。また、α- セルロース分解率は全糖に比べ低くなったもののMacIlvain緩衝液：トルエン＝1.5：8.5から2.5：7.5の割合で前記緩衝液を追加した試験区では分解率がMacIlvain緩衝液のみの試験区より高くなった。低分子の糖ほど親水性になる傾向があることから、単糖のグルコース生産には水分の量が大きく影響したものと考えられた。また、水の分子量18に対し、グルコースの分子量は180と10倍大きい。今回の試験の様に数容量％の水分があれば、およそその10倍のグルコースが得られることになる。通常の植物系のバイオマスは10重量％程度の水分を保有していることから、疎水性の有機溶媒に添加する水分は特に必要とすることなく酵素糖化を行うことが出来る。 Furthermore, the glucose contained in the total sugar was analyzed in the same test. FIG. 8 shows the glucose concentration, and FIG. 9 shows the α-cellulose decomposition rate calculated based on the amount of glucose. The glucose concentration in the extracted water was higher than that in the test group with MacIlvain buffer alone except for the system in which the reaction was carried out only with toluene. In addition, although the degradation rate of α-cellulose was lower than that of total sugar, the degradation rate was only MacIlvain buffer in the test plot where MacIlvain buffer: toluene = 1.5: 8.5 to 2.5: 7.5 was added. It became higher than the ward. It was considered that the amount of water greatly influenced the glucose production of monosaccharides because the sugars with lower molecular weight tend to be more hydrophilic. The molecular weight of glucose is 180, which is 10 times as large as that of water. If there is several percent by volume of water as in this test, about 10 times as much glucose will be obtained. Since ordinary plant-based biomass has about 10% by weight of water, enzymatic saccharification can be performed without the need for water added to a hydrophobic organic solvent.
この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。
本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。
The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.
Claims (5)
疎水性の有機溶媒が存在する反応溶媒中に、バイオマス及び酵素を添加して攪拌することにより、前記バイオマス中の多糖類をより低分子の糖類に加水分解する分解ステップと、
前記分解ステップの最終段階で前記反応溶媒中に抽出用も含め３５容量％以下となるように水系溶媒を添加して、前記分解されたより低分子の糖類を水系溶媒に抽出する抽出ステップと、
前記水系溶媒に抽出された糖類を糖化液として回収する回収ステップと、
を含むことを特徴とする、糖化液を製造する製造方法。 A method for producing a saccharified solution from biomass using an enzyme,
In a reaction solvent in which a hydrophobic organic solvent is present, a biomass and an enzyme are added and stirred to hydrolyze the polysaccharide in the biomass into lower molecular weight saccharides;
An extraction step of extracting by adding the previous SL-aqueous solvent as is a 35% by volume or less, including for extracting the in the reaction solvent at the final stage of decomposition step, the sugars of lower molecular than the decomposed in an aqueous solvent,
A recovery step of recovering the saccharide extracted in the aqueous solvent as a saccharified solution;
A production method for producing a saccharified solution, comprising:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010075425A JP5681923B2 (en) | 2010-03-29 | 2010-03-29 | Method for producing high-concentration saccharified solution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010075425A JP5681923B2 (en) | 2010-03-29 | 2010-03-29 | Method for producing high-concentration saccharified solution |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2011205933A JP2011205933A (en) | 2011-10-20 |
| JP5681923B2 true JP5681923B2 (en) | 2015-03-11 |
Family
ID=44937774
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2010075425A Active JP5681923B2 (en) | 2010-03-29 | 2010-03-29 | Method for producing high-concentration saccharified solution |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5681923B2 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2012359295B2 (en) * | 2012-02-29 | 2015-08-20 | Korea Institute Of Energy Research | High-calorific hybrid coal coated with biomass-derived carbon source, high concentration hybrid coal slurry, and fabrication methods thereof |
| KR101195417B1 (en) * | 2012-03-06 | 2012-10-30 | 한국에너지기술연구원 | Method for manufacturing high-concentration hybrid coal slurry and high-concentration hybrid coal slurry manufactured thereby |
| KR101195416B1 (en) * | 2012-02-29 | 2012-10-29 | 한국에너지기술연구원 | High caloric hybrid coal coated with carbon derived from biomass and manufacturing method thereof |
| KR101195418B1 (en) | 2012-08-08 | 2012-10-30 | 한국에너지기술연구원 | Method for preparation of high caloric hybrid coal coated with carbon derived from biomass using two-step drying process and high caloric hybrid coal prepared thereby |
| KR101210928B1 (en) * | 2012-09-21 | 2012-12-11 | 한국에너지기술연구원 | Method for preparation of glycerol-embedded high caloric hybrid coal and glycerol-embedded high caloric hybrid coal prepared thereby |
| KR20160022191A (en) | 2014-08-19 | 2016-02-29 | 한국에너지기술연구원 | Hyophobic coal-biomass granule type mixed fuel, apparatus and manufacturing method thereof using biomass ethanol process |
| KR20160081027A (en) | 2014-12-30 | 2016-07-08 | 한국에너지기술연구원 | Hydrophobic surfaces coal mixed fuel containing lignin and manufacturing method thereof |
| KR101695702B1 (en) | 2015-07-09 | 2017-01-23 | 한국에너지기술연구원 | MODIFICATION METHOD FOR LOW RANK COAL USING 2nd GENERATION BIOMASS COMPRISED OF HEXOSE |
| KR101709328B1 (en) | 2015-07-09 | 2017-03-08 | 한국에너지기술연구원 | MODIFICATION METHOD FOR LOW RANK COAL USING 2nd GENERATION BIOMASS COMPRISED OF PENTOSE |
| KR101945186B1 (en) | 2016-04-05 | 2019-04-17 | 한국에너지기술연구원 | De-mineralized Biomass, Manufacturing Method and System of Multi-fuel Production thereof |
| KR102028451B1 (en) | 2017-11-02 | 2019-11-14 | 한국에너지기술연구원 | System for high concentration bio-liquid using 2nd generation biomass |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02163091A (en) * | 1988-12-13 | 1990-06-22 | Fuookuueizu Japan:Kk | Production of fructose olilgosaccharide |
| JP2689162B2 (en) * | 1989-02-23 | 1997-12-10 | 株式会社曙ブレーキ中央技術研究所 | Method for enzymatic hydrolysis of cellulose-containing material |
| JP2689161B2 (en) * | 1989-02-23 | 1997-12-10 | 株式会社曙ブレーキ中央技術研究所 | Method for enzymatic hydrolysis of cellulose-containing material |
| JP2873865B2 (en) * | 1990-07-04 | 1999-03-24 | 株式会社曙ブレーキ中央技術研究所 | Method for producing oligosaccharide by enzymatic decomposition of starch-containing material |
| JPH06121693A (en) * | 1992-08-25 | 1994-05-06 | Akebono Brake Res & Dev Center Ltd | Production of oligosaccharide having high polymerization degree |
| JPH0759585A (en) * | 1993-08-20 | 1995-03-07 | Akebono Brake Res & Dev Center Ltd | Production of pullulan oligosaccharide |
| JP2000139490A (en) * | 1998-11-05 | 2000-05-23 | Unitika Ltd | Production of mannose and mannooligosaccharide |
| JP5109121B2 (en) * | 2006-12-28 | 2012-12-26 | 国立大学法人 東京大学 | Method for producing sugar, method for producing ethanol, method for producing lactic acid, cellulose for enzymatic saccharification used therein and method for producing the same |
-
2010
- 2010-03-29 JP JP2010075425A patent/JP5681923B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2011205933A (en) | 2011-10-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5681923B2 (en) | Method for producing high-concentration saccharified solution | |
| Ezeilo et al. | Raw oil palm frond leaves as cost-effective substrate for cellulase and xylanase productions by Trichoderma asperellum UC1 under solid-state fermentation | |
| JP6356292B2 (en) | Biomass processing method | |
| Pensupa et al. | A solid state fungal fermentation-based strategy for the hydrolysis of wheat straw | |
| Lee et al. | Chemo-enzymatic saccharification and bioethanol fermentation of lipid-extracted residual biomass of the microalga, Dunaliella tertiolecta | |
| Dai et al. | Combination of biological pretreatment with NaOH/Urea pretreatment at cold temperature to enhance enzymatic hydrolysis of rice straw | |
| Chen et al. | Effects of laccase on lignin depolymerization and enzymatic hydrolysis of ensiled corn stover | |
| JP5752047B2 (en) | Organic solvent pretreatment of biomass to promote enzymatic saccharification | |
| Maitan-Alfenas et al. | The influence of pretreatment methods on saccharification of sugarcane bagasse by an enzyme extract from Chrysoporthe cubensis and commercial cocktails: a comparative study | |
| Hsu et al. | Pretreatment and hydrolysis of cellulosic agricultural wastes with a cellulase-producing Streptomyces for bioethanol production | |
| US8911976B2 (en) | Lignin-based enzyme stabilizer | |
| JP2012512658A (en) | Organic solvent pretreatment of biomass to promote enzymatic saccharification | |
| Outeirino et al. | A novel approach to the biorefinery of brewery spent grain | |
| Lienqueo et al. | Second generation bioethanol from Eucalyptus globulus Labill and Nothofagus pumilio: ionic liquid pretreatment boosts the yields | |
| JP2011092151A (en) | Method of processing vegetable raw material | |
| Radhakumari et al. | Pongamia pinnata seed residue–A low cost inedible resource for on-site/in-house lignocellulases and sustainable ethanol production | |
| Sheetal et al. | Effect of cultivar variation and Pichia stipitis NCIM 3498 on cellulosic ethanol production from rice straw | |
| Chang et al. | Improving the remaining activity of lignocellulolytic enzymes by membrane entrapment | |
| Rajesh et al. | Production of multienzymes, bioethanol, and acetic acid by novel Bacillus sp. PM06 from various lignocellulosic biomass | |
| Smichi et al. | Pretreatment and enzymatic saccharification of new phytoresource for bioethanol production from halophyte species | |
| Triwahyuni | Valorization of oil palm empty fruit bunch for bioethanol production through separate hydrolysis and fermentation (SHF) using immobilized cellulolytic enzymes | |
| Rajeswari et al. | A concerted enzymatic de-structuring of lignocellulosic materials using a compost-derived microbial consortia favoring the consolidated pretreatment and bio-saccharification | |
| JP5824074B2 (en) | Bioethanol production method and system | |
| Elgharbawy et al. | Implementation of definite screening design in optimization of in situ hydrolysis of EFB in cholinium acetate and locally produced cellulase combined system | |
| Gajula et al. | Fermentation of groundnut shell enzymatic hydrolysate for fuel ethanol production by free and sorghum stalks immobilized cells of Pichia stipitis NCIM 3498 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20130215 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20140624 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20140822 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20141126 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20141205 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 5681923 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |