JP2007074993A - Method for saccharifying biomass containing hemicellulose and saccharide given by the same - Google Patents
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Abstract
Description
本発明は、小片状の木片の酵素処理による糖化方法及びそれによって得られた糖に関し、更に詳しくは木片に含まれるヘミセルロースを酵素を使って処理する前段階として過酸化水素水により熱水処理を行うヘミセルロースの糖化方法に関し、またその糖化方法によって得られた糖に関する。 The present invention relates to a saccharification method by enzymatic treatment of small pieces of wood pieces and the sugar obtained thereby, and more specifically, hydrothermal treatment with hydrogen peroxide as a pre-stage for treating hemicellulose contained in wood pieces with enzymes. The present invention relates to a method for saccharification of hemicellulose, and to a saccharide obtained by the saccharification method.
木材チップ、木片、稲ワラ、籾殻等のいわゆるバイオマスを有効利用する技術が開発されている。
これらバイオマスの成分であるヘミセルロースは廃材の中に存在するため非常に安価でもある。
このヘミセルロースを出発原料として種々の物質を生み出す技術が求められている。
中でも最近のアルコール発酵技術の需要にともない、バイオマスから糖(キシロオリゴ糖、セロオリゴ糖、キシロースなど)を製造する技術の開発がされている。
Technologies for effectively utilizing so-called biomass such as wood chips, wood chips, rice straw and rice husks have been developed.
Since hemicellulose, which is a component of these biomasses, is present in the waste material, it is also very inexpensive.
There is a need for a technique for producing various substances using hemicellulose as a starting material.
In particular, with the recent demand for alcohol fermentation technology, technology for producing sugar (xylooligosaccharide, cellooligosaccharide, xylose, etc.) from biomass has been developed.
それら技術のうちに、例えばヘミセルロースを糖化する方法としては無水酢酸や濃硫酸を使う方法がある。
しかし上記の方法では乾燥工程を必要とし、またヘミセルロースの加水分解も激しく行われるため反応系の設備が複雑となる。
さらに設備内では酸による腐食も起きやすく、また硫酸回収にも余分なコストが発生する。
これらの問題を解決する手段として緩和な条件で加水分解を行うことができる酵素学的方法が開発されている。
Among these techniques, for example, as a method for saccharifying hemicellulose, there is a method using acetic anhydride or concentrated sulfuric acid.
However, the above method requires a drying step, and the hydrolysis of hemicellulose is also performed vigorously, so that the equipment of the reaction system becomes complicated.
Furthermore, corrosion due to acid is likely to occur in the facility, and extra cost is required for sulfuric acid recovery.
Enzymatic methods capable of performing hydrolysis under mild conditions have been developed as means for solving these problems.
この方法は酵素(例えばヘミセルラーゼ)を使って加水分解するもので、この方法を用いると装置に耐圧性、耐酸性の反応容器を必要としないだけでなく環境汚染も少ない。
このような酵素を使った糖化方法としては例えば特許文献1、特許文献2等がある。
This method hydrolyzes using an enzyme (eg, hemicellulase). When this method is used, the apparatus does not require a pressure-resistant and acid-resistant reaction vessel, and environmental pollution is small.
Examples of saccharification methods using such enzymes include Patent Document 1 and Patent Document 2.
しかしながら一般にヘミセルロースにはリグニンが糊着していて、このリグニンの存在が糖化の大きな障害となっている。
このリグニンのためにヘミセルロースの加水分解効率が大きく低下し、糖化率を極めて低いものとしている。
そこで、リグニンを分解し除去する発明を以前我々はオゾン処理を行うことで達成した(特願2004−333445)。
しかし、この発明はバイオマスがよく粉砕された粒状である場合は非常に有効であるが、ある程度の大きさ(大きさ1cm〜10cm程度)の小片状の木材に対してはその効果が有効に発揮されない。
また、酵素糖化に用いる酵素は非常に高価であるので酵素の使用は製造コストを増大させてしまう。
However, lignin is generally glued to hemicellulose, and the presence of this lignin is a major obstacle to saccharification.
Due to this lignin, the hydrolysis efficiency of hemicellulose is greatly reduced, and the saccharification rate is extremely low.
Thus, the invention of decomposing and removing lignin was previously achieved by performing ozone treatment (Japanese Patent Application No. 2004-333445).
However, this invention is very effective when the biomass is in the form of finely pulverized granules, but the effect is effective for small pieces of wood of a certain size (size of about 1 cm to 10 cm). It is not demonstrated.
Moreover, since the enzyme used for enzymatic saccharification is very expensive, the use of the enzyme increases the production cost.
本発明はこのような技術的背景をもとになされたものである。
すなわち本発明は、セルロースを含むバイオマスが細かい粒状でなくても効率よく加水分解でき、糖の収率も高いセルロースの糖化方法を提供するものである。
またその糖化方法によって得られた糖を提供するものである。
The present invention has been made based on such a technical background.
That is, the present invention provides a method for saccharification of cellulose that can be efficiently hydrolyzed even if the biomass containing cellulose is not fine and has a high sugar yield.
Moreover, the sugar obtained by the saccharification method is provided.
本発明者は上記問題点について鋭意研究を行った結果、酵素による糖化処理以前に過酸化水素水で熱水処理をすることで、酵素による糖化処理の障害となるリグニンのスムーズな分解、除去が可能になることを見出した。 As a result of diligent research on the above problems, the present inventor has performed hydrothermal treatment with hydrogen peroxide before the saccharification treatment with the enzyme, thereby smoothly decomposing and removing lignin that hinders the saccharification treatment with the enzyme. I found it possible.
すなわち本発明は、(1)、バイオマスに含まれるヘミセルロースを酵素を使って加水分解し糖化する方法であって、酵素を使って加水分解する前段階で、過酸化水素水を使った熱水処理を行って、そこに含まれるリグニンを分解、除去させるヘミセルロースの糖化方法に存する。 That is, the present invention is (1) a method for hydrolyzing and saccharifying hemicellulose contained in biomass by using an enzyme, and hydrothermal treatment using hydrogen peroxide water in a stage prior to hydrolysis using the enzyme. To saccharify hemicellulose by decomposing and removing lignin contained therein.
また本発明は、(2)、ヘミセルロースを加水分解し、糖を得るための酵素としてヘミセルラーゼを使用する上記(1)記載のヘミセルロースの糖化方法に存する。 Moreover, this invention exists in the saccharification method of the hemicellulose of the said (1) description which uses hemicellulase as an enzyme for hydrolyzing hemicellulose and obtaining saccharide | sugar.
また本発明は、(3)、上記バイオマスが小片状である(1)記載のヘミセルロースの糖化方法に存する。 The present invention also resides in (3) the method for saccharification of hemicellulose according to (1), wherein the biomass is in the form of small pieces.
また本発明は、(4)、上記(1)〜(3)の少なくとも1項記載の糖化方法によって製造された糖に存する。 Moreover, this invention exists in the saccharide | sugar manufactured by the saccharification method of at least 1 description of (4) and said (1)-(3).
また本発明は、(5)、製造された糖がアラビノース、ガラクトース、マンノース、キシロース、キシロオリゴ糖、グルコースである(4)記載の糖に存する。 Further, the present invention resides in (5) the sugar according to (4), wherein the produced sugar is arabinose, galactose, mannose, xylose, xylooligosaccharide, glucose.
なお、本発明の目的に添ったものであれば、上記の発明を適宜組み合わせた構成も採用可能である。 In addition, as long as the objective of this invention is met, the structure which combined said invention suitably is also employable.
ヘミセルロースを酵素糖化の前に予め過酸化水素水で熱水処理しておくことで、ヘミセルロースに糊着していたリグニンが分解、除去された状態で加水分解されるため非常に効率よく単糖(アラビノース、ガラクトース、マンノース等)を回収することができるようになる。
また、同じリグニン分解効果のあるオゾン処理と異なり細かい粒状のバイオマスでなく、小片状のバイオマスであっても十分なリグニン分解効果を発揮できる。
これはバイオマスを小片状から粒状に粉砕するエネルギーが不要となるので単糖生成におけるエネルギー効率の改善に非常に効果的である。
Hemicellulose is hydrothermally treated with hydrogen peroxide in advance before enzymatic saccharification, so that the lignin glued to hemicellulose is hydrolyzed in a state where it is decomposed and removed. Arabinose, galactose, mannose, etc.) can be recovered.
Moreover, unlike the ozone treatment with the same lignin decomposition effect, even if it is not a fine granular biomass but small piece biomass, sufficient lignin decomposition effect can be exhibited.
This eliminates the need for energy to pulverize biomass from small pieces to granules, and is very effective in improving energy efficiency in monosaccharide production.
また、過酸化水素水で熱水処理を行うと酵素を用いなくとも同程度の単糖を回収することができる。
酵素は一般的に高価であるのでこのことは単糖製造コストの削減に非常に効果的である。
In addition, when hydrothermal treatment is performed with hydrogen peroxide, the same level of monosaccharide can be recovered without using an enzyme.
Since enzymes are generally expensive, this is very effective in reducing monosaccharide production costs.
本発明はヘミセルロースを、酵素を使って分解し、糖化する過程において最初に過酸化水素水で熱水処理を施すことでリグニンを分解させることに大きな特徴がある。
以下図1のフロー図に従って糖化工程を説明する。
The present invention is greatly characterized in that lignin is decomposed by first performing hydrothermal treatment with hydrogen peroxide in the process of decomposing and saccharifying hemicellulose using an enzyme.
Hereinafter, the saccharification process will be described with reference to the flowchart of FIG.
〔過酸化水素水による熱水処理〕
バイオマス内に存在しているヘミセルロースにはリグニンが糊着している。
この状態ではヘミセルロースを可溶化し、酵素糖化を行おうとしても単糖の収率が悪い。
そのため過酸化水素水を用いて高温で一定時間バイオマスを加熱する熱水処理を行うことでリグニンが分解され、ヘミセルロースからリグニンが容易に溶脱される。
このときリグニンは没食子酸、シュウ酸、酢酸、ギ酸等の有機酸になり水に溶解する。
なお熱水処理の際にバイオマスを粒状にまで細かくする必要は無くある程度の大きさ(長さ1cm〜10cm)を持った小片状であっても十分にリグニンを溶脱する効果を発揮する。
熱水処理を行う場合は通常、温度は140℃〜160℃、処理時間は2時間〜4時間で行うことが好ましい。
[Hot water treatment with hydrogen peroxide]
Lignin is glued to the hemicellulose present in the biomass.
In this state, the yield of monosaccharides is poor even if hemicellulose is solubilized and enzymatic saccharification is attempted.
Therefore, lignin is decomposed by performing a hydrothermal treatment in which biomass is heated at a high temperature for a certain period of time using hydrogen peroxide, and lignin is easily leached from hemicellulose.
At this time, lignin becomes an organic acid such as gallic acid, oxalic acid, acetic acid, and formic acid and dissolves in water.
In addition, it is not necessary to make biomass into a granular form at the time of hydrothermal treatment, and even if it is a small piece having a certain size (length: 1 cm to 10 cm), the effect of sufficiently leaching lignin is exhibited.
In the case of performing the hot water treatment, it is usually preferred that the temperature is 140 ° C. to 160 ° C., and the treatment time is 2 hours to 4 hours.
上記熱水処理を行うことで、本来では水に溶けないヘミセルロースを可溶化することができる。
すなわち我々の以前の発明(特願2004−333445)ではリグニンをオゾン処理で可溶化させた後、ヘミセルロースを可溶化させる、という二つの工程があったが、これらが一つにまとまり作業効率、エネルギー効率の両面で有効となった。
By performing the hot water treatment, hemicellulose that is not originally soluble in water can be solubilized.
That is, in our previous invention (Japanese Patent Application No. 2004-333445), there were two steps of solubilizing lignin by ozone treatment and then solubilizing hemicellulose. Effective in terms of both efficiency.
〔オゾン処理〕
オゾン処理を行う場合は熱水処理後の溶液を蒸留水で満たした容器に懸濁させて、オゾンガスを散気管方式により該容器に注入し1〜3時間処理を行う。
[Ozone treatment]
In the case of performing ozone treatment, the solution after the hot water treatment is suspended in a container filled with distilled water, and ozone gas is injected into the container by an air diffuser method to perform the treatment for 1 to 3 hours.
〔ヘミセルロースの酵素糖化〕
熱水処理(又は熱水処理とオゾン処理)済みの溶液に適量の酵素を添加し、十分加水分解させるためにある適当な温度の下で一定時間放置し、溶液を濾過してキシロオリゴ糖及び/又はキシロースを得る。
ここでヘミセルロースを加水分解する酵素としては、キシラナーゼ、ヘミセルラーゼ、セルラーゼ等を適宣、単数又は複数選択して採用する。
酵素による加水分解させる際の条件は酵素を最も活性化させるという観点から40℃〜60℃、pH3〜4、24時間以上反応させることが好ましい。
[Enzymatic saccharification of hemicellulose]
Add an appropriate amount of enzyme to the hydrothermally treated solution (or hydrothermally treated and ozone treated), leave it at a suitable temperature for a certain period of time to fully hydrolyze it, filter the solution, and xylo-oligosaccharide and / or Alternatively, xylose is obtained.
Here, as the enzyme that hydrolyzes hemicellulose, xylanase, hemicellulase, cellulase and the like are suitably selected and used.
The conditions for the hydrolysis with the enzyme are preferably 40 to 60 ° C., pH 3 to 4 for 24 hours or more from the viewpoint of activating the enzyme most.
以下、本発明を実施例を挙げて説明するが本発明は実施例に限らず種々の変形例が可能である。 Hereinafter, the present invention will be described with reference to examples. However, the present invention is not limited to the examples, and various modifications are possible.
〔実施例1〕
まず試料として木片(本実験では杉を使用)を2cm角程度の小片状にしたものを使用する。
次に試料2.5g、過酸化水素水(31重量%)2mlと水を合わせて40mlの試験溶液作り、これを水熱反応容器(内容積50ml、ハステロイ製)に入れ150℃、4時間で熱水処理を行う。
上記熱水処理済みの溶液を濾過し、ろ液に加水分解酵素としてヘミセルラーゼ(セルロシンH100:HBI社商品名)をろ液5mlに対して3mgの割合で添加して酵素糖化を行う。
酵素糖化条件はpH4、温度50℃、反応時間24時間とした。
単糖の分析はイオンクロマトグラフ(DIONEX社製)で行った。
実験の結果ヘミセルロース由来の単糖(アラビノース、ガラクトース、マンノース、キシロース、キシロオリゴ糖)及びグルコースが検出された。
[Example 1]
First, as a sample, a piece of wood (cedar used in this experiment) in the shape of a small piece of about 2 cm square is used.
Next, 2.5 g of the sample, 2 ml of hydrogen peroxide solution (31 wt%) and water were combined to make a 40 ml test solution, which was placed in a hydrothermal reaction vessel (internal volume 50 ml, made by Hastelloy) at 150 ° C. for 4 hours. Perform hot water treatment.
The hydrothermally treated solution is filtered, and hemicellulase (Cellulosin H100: trade name of HBI) as a hydrolase is added to the filtrate at a rate of 3 mg with respect to 5 ml of the filtrate to perform enzymatic saccharification.
The enzymatic saccharification conditions were pH 4, temperature 50 ° C., and reaction time 24 hours.
The analysis of monosaccharides was performed with an ion chromatograph (manufactured by DIONEX).
As a result of the experiment, hemicellulose-derived monosaccharides (arabinose, galactose, mannose, xylose, xylooligosaccharide) and glucose were detected.
〔実施例2〕
酵素糖化しないこと以外は全て実施例1と同様の手順で行った。
[Example 2]
All procedures were the same as in Example 1 except that enzymatic saccharification was not performed.
〔実施例3〕
熱水処理後にオゾン処理を2時間行ったこと以外は、全て実施例1と同様の手順で行った。
Example 3
All procedures were the same as in Example 1 except that ozone treatment was performed for 2 hours after the hot water treatment.
〔実施例4〕
酵素糖化しないこと以外は全て実施例3と同様の手順で行った。
Example 4
All procedures were the same as in Example 3 except that enzymatic saccharification was not performed.
〔比較例1〕
熱水処理を行わず、試料2.5gを蒸留水で満たした容器に懸濁させて、オゾンガスを散気管方式によりオゾン処理したこと以外は全て実施例1と同様の手順で行った。
[Comparative Example 1]
The procedure was the same as in Example 1 except that 2.5 g of the sample was suspended in a container filled with distilled water without performing the hot water treatment, and ozone gas was ozone treated by an air diffuser method.
〔比較例2〕
酵素糖化しないこと以外は全て比較例1と同様の手順で行った。
[Comparative Example 2]
All procedures were the same as those in Comparative Example 1 except that enzymatic saccharification was not performed.
〔比較例3〕
オゾン処理をしないこと以外は全て比較例1と同様の手順で行った。
[Comparative Example 3]
All procedures were the same as in Comparative Example 1 except that the ozone treatment was not performed.
〔比較例4〕
酵素糖化しないこと以外は全て比較例3と同様の手順で行った。
以上実施例1〜4、比較例1〜4の結果回収されたグルコース以外の単糖(アラビノース、ガラクトース、マンノース、キシロース、キシロオリゴ糖)の量を試料1gあたりの回収量に換算して表1に示す。
[Comparative Example 4]
All procedures were the same as in Comparative Example 3 except that enzymatic saccharification was not performed.
Table 1 shows the amount of monosaccharides other than glucose (arabinose, galactose, mannose, xylose, xylooligosaccharide) recovered as a result of Examples 1 to 4 and Comparative Examples 1 to 4 converted to the recovered amount per 1 g of sample. Show.
表1より過酸化水素水による熱水処理はオゾン処理よりもリグニンを分解する効果が高くグルコースを大量に回収することができた。
さらに過酸化水素による熱水処理を行い酵素処理を行わない場合(実施例2)における単糖回収量は、過酸化水素による熱水処理と酵素処理を行う場合(実施例1)における単糖回収量の約92%(109mg/118mg)を回収することができた。
この回収量はオゾン処理と酵素処理(比較例1)を行う場合における単糖回収量を上回っており効果としては必要且つ十分であるといえる。
From Table 1, the hydrothermal treatment with hydrogen peroxide was more effective in decomposing lignin than the ozone treatment, and a large amount of glucose could be recovered.
Furthermore, the amount of monosaccharides recovered when hydrothermal treatment with hydrogen peroxide is performed and no enzyme treatment is performed (Example 2) is the amount of monosaccharides recovered when hydrothermal treatment with hydrogen peroxide and enzyme treatment are performed (Example 1). Approximately 92% of the amount (109 mg / 118 mg) could be recovered.
This recovery amount exceeds the recovery amount of monosaccharides in the case of performing ozone treatment and enzyme treatment (Comparative Example 1), and can be said to be necessary and sufficient as an effect.
〔表1〕
[Table 1]
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US8618280B2 (en) | 2009-11-30 | 2013-12-31 | Applied Biorefinery Sciences Llc | Biorefinery process for extraction, separation, and recovery of fermentable saccharides, other useful compounds, and yield of improved lignocellulosic material from plant biomass |
JP2012161292A (en) * | 2011-02-08 | 2012-08-30 | Unitika Ltd | Method for producing composition containing monosaccharide |
CN103044497A (en) * | 2013-01-12 | 2013-04-17 | 山东绿健生物技术有限公司 | Preparation method of moisture-resistant xylooligosaccharide powder |
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