JP2012223113A - Method for saccharifying biomass - Google Patents
Method for saccharifying biomass Download PDFInfo
- Publication number
- JP2012223113A JP2012223113A JP2011092374A JP2011092374A JP2012223113A JP 2012223113 A JP2012223113 A JP 2012223113A JP 2011092374 A JP2011092374 A JP 2011092374A JP 2011092374 A JP2011092374 A JP 2011092374A JP 2012223113 A JP2012223113 A JP 2012223113A
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- JP
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- Prior art keywords
- enzyme
- saccharification
- solution
- recovery
- reaction
- 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.)
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- KHILLYASAGTPOI-UHFFFAOYSA-M sodium;9,10-dioxoanthracene-2-sulfonate;hydrate Chemical compound O.[Na+].C1=CC=C2C(=O)C3=CC(S(=O)(=O)[O-])=CC=C3C(=O)C2=C1 KHILLYASAGTPOI-UHFFFAOYSA-M 0.000 description 1
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- 239000001384 succinic acid Substances 0.000 description 1
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- 108010083879 xyloglucan endo(1-4)-beta-D-glucanase Proteins 0.000 description 1
- 235000020138 yakult Nutrition 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、バイオマスの糖化方法に関するものであり、より詳細には、リグノセルロース系バイオマスを酵素で糖化する方法に関するものである。 The present invention relates to a biomass saccharification method, and more particularly to a method of saccharifying lignocellulosic biomass with an enzyme.
リグノセルロース系バイオマスを糖化し、発酵原料となる単糖類を得る技術は、食料と競合しない非可食性のバイオマスの資源・エネルギー利用という観点から、極めて重要な技術である。リグノセルロース系バイオマスを糖化する方法は、硫酸等の酸を用いて加水分解する酸糖化法と、酵素を用いて加水分解する酵素糖化法に大別される。酸糖化法はバイオマスとの反応性が高いという利点があるが、耐酸性の反応器を必要とし、使用後の酸を中和・回収する工程が必要となる点で課題を有する。一方、酵素糖化法は、比較的マイルドな反応条件で分解反応が進行するので、酸糖化法と比較して設備コストが低い、あるいは反応時における安全性が高いという利点を有する。しかしながら、酵素は非常に高価であり、酵素コストが実用上の大きな課題となっている。 The technology for saccharifying lignocellulosic biomass to obtain monosaccharides as fermentation raw materials is an extremely important technology from the viewpoint of the use of non-edible biomass that does not compete with food and energy. Methods for saccharifying lignocellulosic biomass are broadly classified into acid saccharification methods in which hydrolysis is performed using an acid such as sulfuric acid, and enzyme saccharification methods in which hydrolysis is performed using an enzyme. The acid saccharification method has an advantage of high reactivity with biomass, but has a problem in that it requires an acid-resistant reactor and a step of neutralizing and recovering the acid after use. On the other hand, the enzymatic saccharification method has an advantage that the equipment costs are lower or the safety during the reaction is higher than the acid saccharification method because the decomposition reaction proceeds under relatively mild reaction conditions. However, the enzyme is very expensive, and the enzyme cost is a big problem in practical use.
酵素コストを低減する方策として酵素リサイクルが試みられているが、問題となるのは糖化反応残渣（糖化反応後の残渣）への酵素の吸着である。すなわち、リグノセルロース系バイオマスを糖化するために使用される酵素は、未分解の糖質およびリグニンに対して高い吸着性を示すため、糖化反応残渣に吸着する。この吸着現象が、酵素をリサイクルする上で大きな課題となっている（非特許文献１参照）。一方、酵素処理後の糖化反応液からの酵素の回収は、一般に膜分離法（限外ろ過）、またはリグノセルロース系バイオマスへの再吸着により行えることが知られており、比較的高い回収率が見込める。 Enzyme recycling has been attempted as a measure for reducing the enzyme cost, but the problem is the adsorption of the enzyme to the saccharification reaction residue (residue after the saccharification reaction). That is, the enzyme used for saccharifying lignocellulosic biomass exhibits high adsorptivity to undegraded carbohydrates and lignin, and therefore adsorbs to saccharification reaction residues. This adsorption phenomenon is a major problem in recycling the enzyme (see Non-Patent Document 1). On the other hand, it is known that enzyme recovery from saccharification reaction liquid after enzyme treatment is generally performed by membrane separation method (ultrafiltration) or re-adsorption to lignocellulosic biomass. I can expect.
糖化反応残渣に吸着した酵素の回収・リサイクル方法としては、例えば、以下の（ａ）〜（ｄ）などが知られている。
（ａ）酵素が吸着した糖化反応残渣をそのまま次回の酵素糖化反応に再利用する方法（特許文献１、非特許文献２参照）
（ｂ）界面活性剤を使用して糖化反応残渣から酵素を脱着・回収する方法（非特許文献３参照）
（ｃ）アルカリで糖化反応残渣から酵素を脱着・回収する方法（非特許文献３、４、５参照）
（ｄ）酸性〜中性の高濃度のバッファーで糖化反応残渣から酵素を脱着・回収する方法（非特許文献６、７参照）
As methods for recovering and recycling the enzyme adsorbed on the saccharification reaction residue, for example, the following (a) to (d) are known.
(A) A method of reusing a saccharification reaction residue adsorbed with an enzyme as it is for the next enzyme saccharification reaction (see Patent Document 1 and Non-Patent Document 2)
(B) A method of desorbing and recovering an enzyme from a saccharification reaction residue using a surfactant (see Non-Patent Document 3)
(C) Method for desorbing and recovering enzyme from saccharification reaction residue with alkali (see Non-Patent Documents 3, 4 and 5)
(D) A method for desorbing and recovering an enzyme from a saccharification reaction residue with an acidic to neutral high-concentration buffer (see Non-Patent Documents 6 and 7).
上記（ａ）の方法は、リグニンを多く含むリグノセルロース系バイオマスを原料とする場合は、リグニンが蓄積するため適用することができない。一般に、リグニンを除去するには多大のコスト（多量のアルカリや酸等）を必要とするため、リグニン含量の高いリグノセルロース系バイオマスに適用可能な低コストのリサイクル方法が望まれる。
上記（ｂ）の方法は、酵素脱着効果が十分でなく酵素回収率が低いこと、界面活性剤を使用するためコスト高になることなどの課題を有している。
上記（ｃ）の方法は、アルカリによる酵素の失活が課題であり、また、高い回収率で酵素を回収した例は知られていない。
上記（ｄ）の方法は、高濃度のバッファー（例えば、０．５Ｍリン酸バッファー）を大量に使用するため、コスト高になるという課題を有している。
The method (a) cannot be applied when lignocellulosic biomass containing a large amount of lignin is used as a raw material because lignin accumulates. In general, a large amount of cost (a large amount of alkali, acid, etc.) is required to remove lignin. Therefore, a low-cost recycling method applicable to lignocellulosic biomass having a high lignin content is desired.
The method (b) has problems such that the enzyme desorption effect is not sufficient, the enzyme recovery rate is low, and the cost is high because a surfactant is used.
In the method (c), the inactivation of the enzyme by alkali is a problem, and no example of recovering the enzyme at a high recovery rate is known.
The method (d) has a problem of high cost because a large amount of a high concentration buffer (for example, 0.5 M phosphate buffer) is used.
また、リグノセルロースはセルロースのみでなくヘミセルロースも含むため、糖化酵素としてセルラーゼとヘミセルラーゼを同時に作用させ、かつ、同時にリサイクルできることが望ましい。しかし、上記（ａ）〜（ｄ）の方法は、セルラーゼの回収・リサイクルに適用されており、ヘミセルラーゼ、またはセルラーゼとヘミセルラーゼの混合物に関する回収・リサイクルの報告はない。 Further, since lignocellulose contains not only cellulose but also hemicellulose, it is desirable that cellulase and hemicellulase simultaneously act as saccharifying enzymes and can be recycled at the same time. However, the methods (a) to (d) are applied to cellulase recovery / recycling, and there is no report of recovery / recycling regarding hemicellulase or a mixture of cellulase and hemicellulase.
本発明は、リグニン含量の高いリグノセルロース系バイオマスに適用でき、糖化に用いた酵素を高い回収率で回収可能な酵素回収工程を含むリグノセルロース系バイオマスの糖化方法を提供することを課題とする。 An object of the present invention is to provide a method for saccharification of lignocellulosic biomass, which can be applied to lignocellulosic biomass having a high lignin content and includes an enzyme recovery step capable of recovering an enzyme used for saccharification at a high recovery rate.
本発明は、上記課題を解決するために、以下の発明を包含する。
［１］リグノセルロース系バイオマスを酵素で糖化する糖化工程と、糖化工程終了後に酵素を回収する酵素回収工程を含むリグノセルロース系バイオマスの糖化方法であって、
酵素回収工程が、以下の（Ａ）および（Ｂ）の工程のうち少なくとも１つを含むことを特徴する糖化方法。
（Ａ）糖化反応スラリーにアルカリを添加して、糖化反応残渣に吸着した酵素を脱着させた後、糖化反応スラリーの固液分離を行って酵素を含有する酵素回収液を回収する工程
（Ｂ）糖化反応スラリーの固液分離により得られた糖化反応残渣にアルカリを添加して、糖化反応残渣に吸着した酵素を脱着させて酵素を含有する酵素回収液を回収し、その際、酵素回収液のｐＨが漸増するように糖化反応残渣にアルカリを添加する工程
［２］糖化工程の前に、リグノセルロース系バイオマスの酵素糖化効率を高める処理を行う前処理工程を有することを特徴とする前記［１］に記載の糖化方法。
［３］酵素がセルラーゼおよびヘミセルラーゼの混合物である前記［１］または［２］に記載の糖化方法。
［４］セルラーゼが少なくともセロビオヒドロラーゼ、β−グルカナーゼおよびβ−グルコシダーゼを含み、ヘミセルラーゼが少なくともキシラナーゼおよびβ−キシロシダーゼを含むことを特徴とする前記［３］に記載の糖化方法。
［５］（Ｂ）の工程において、ｐＨ漸増の終点における酵素回収液のｐＨが７〜１３の範囲内であることを特徴とする前記［１］〜［４］のいずれかに記載の糖化方法。
［６］酵素回収工程において、酵素回収液を回収後速やかに、酵素回収液のｐＨを弱酸性〜中性に調整することを特徴とする前記［１］〜［５］のいずれかに記載の糖化方法。
［７］前処理工程が、リグノセルロース系バイオマスをアルカリ処理するものであり、その際に生じたアルカリ廃液を、酵素回収工程のアルカリとして用いることを特徴とする前記［２］〜［６］のいずれかに記載の糖化方法。
The present invention includes the following inventions in order to solve the above problems.
[1] A saccharification method for lignocellulosic biomass comprising a saccharification step for saccharifying lignocellulosic biomass with an enzyme, and an enzyme recovery step for recovering the enzyme after completion of the saccharification step,
The saccharification method, wherein the enzyme recovery step includes at least one of the following steps (A) and (B).
(A) Step (B) of adding an alkali to the saccharification reaction slurry to desorb the enzyme adsorbed on the saccharification reaction residue and then performing solid-liquid separation of the saccharification reaction slurry to recover an enzyme recovery solution containing the enzyme. An alkali is added to the saccharification reaction residue obtained by solid-liquid separation of the saccharification reaction slurry, and the enzyme adsorbed on the saccharification reaction residue is desorbed to recover the enzyme recovery solution containing the enzyme. The step [2] of adding an alkali to a saccharification reaction residue so that the pH gradually increases [2] The saccharification step has a pretreatment step of performing a treatment for increasing the enzymatic saccharification efficiency of lignocellulosic biomass [1] ] The saccharification method of description.
[3] The saccharification method according to the above [1] or [2], wherein the enzyme is a mixture of cellulase and hemicellulase.
[4] The saccharification method according to [3], wherein the cellulase contains at least cellobiohydrolase, β-glucanase and β-glucosidase, and the hemicellulase contains at least xylanase and β-xylosidase.
[5] The saccharification method according to any one of [1] to [4], wherein in the step (B), the pH of the enzyme recovery solution at the end of the gradual increase in pH is in the range of 7 to 13. .
[6] The enzyme recovery step according to any one of [1] to [5], wherein in the enzyme recovery step, the pH of the enzyme recovery solution is adjusted to slightly acidic to neutral immediately after recovering the enzyme recovery solution. Saccharification method.
[7] The above-mentioned [2] to [6], wherein the pretreatment step is an alkali treatment of lignocellulosic biomass, and the alkaline waste liquid generated at that time is used as an alkali in the enzyme recovery step. The saccharification method in any one.
本発明によれば、リグニン含量の高いリグノセルロース系バイオマスに適用でき、糖化に用いた酵素を高い回収率で回収可能な酵素回収工程を含むリグノセルロース系バイオマスの糖化方法を提供することができる。回収した酵素をリサイクル使用して糖化工程を行うことにより、酵素使用量を減らし、酵素コストを大幅に低減することが可能となる。 ADVANTAGE OF THE INVENTION According to this invention, it can apply to lignocellulosic biomass with a high lignin content, and can provide the saccharification method of lignocellulosic biomass including the enzyme collection | recovery process which can collect | recover the enzyme used for saccharification with a high recovery rate. By recycling the collected enzyme and performing the saccharification step, the amount of enzyme used can be reduced, and the enzyme cost can be greatly reduced.
本発明は、リグノセルロース系バイオマスの糖化方法を提供する。本発明の糖化方法は、リグノセルロース系バイオマスを酵素で糖化する糖化工程と、糖化工程終了後に酵素を回収する酵素回収工程を必須の工程として含むものであればよい。好ましくは、糖化工程の前に、リグノセルロース系バイオマスの酵素糖化効率を高める処理を行う前処理工程を含む。 The present invention provides a method for saccharification of lignocellulosic biomass. The saccharification method of this invention should just contain the saccharification process which saccharifies lignocellulosic biomass with an enzyme, and the enzyme recovery process which collect | recovers an enzyme after completion | finish of a saccharification process as an essential process. Preferably, before the saccharification step, a pretreatment step of performing a treatment for increasing the enzymatic saccharification efficiency of lignocellulosic biomass is included.
本発明の糖化方法の原料は、リグノセルロース系バイオマスを含むものであればよい。リグノセルロース系バイオマスは、主としてセルロース、ヘミセルロースおよびリグニンから構成されており、木本植物、草本植物、それらの加工品、それらの廃棄物などが該当する。具体的には、例えば、木材、間伐材、製材残材、建築廃材、樹皮、果房、果実殻、茎葉、稲わら、麦わら、バガス、古紙などが挙げられる。好ましくは、アブラヤシ、ナツメヤシ、サゴヤシ、ココヤシ等のヤシ類（幹、茎葉、空果房、果実繊維）、サトウキビ（バガス、葉）、トウモロコシ（穂軸、茎葉）、稲わら、麦わら、スウィッチグラス、および、ユーカリ、ポプラ、スギ等の木材（樹皮、木部）である。より好ましくはヤシ類の空果房、サトウキビバガス、トウモロコシ穂軸、稲わら、麦わら、ユーカリ、スギであり、さらに好ましくはアブラヤシの空果房である。リグノセルロース系バイオマスの大きさ、形状等は特に限定されないが、各工程における反応効率を向上させる観点から、予め粉砕され、チップ状にされたものが好ましい。 The raw material of the saccharification method of this invention should just contain lignocellulosic biomass. Lignocellulosic biomass is mainly composed of cellulose, hemicellulose, and lignin, and includes woody plants, herbaceous plants, processed products thereof, wastes thereof, and the like. Specific examples include timber, thinned wood, sawn timber, building waste, bark, fruit bunches, fruit shells, foliage, rice straw, wheat straw, bagasse, and waste paper. Preferably, palms such as oil palm, date palm, sago palm, and coconut palm (stem, foliage, empty fruit bunch, fruit fiber), sugar cane (bagasse, foliage), corn (cob, foliage), rice straw, straw, switchgrass, And wood (bark, xylem) such as eucalyptus, poplar and cedar. More preferred are empty fruit bunches of palm, sugarcane bagasse, corn cob, rice straw, straw, eucalyptus and cedar, and more preferred are empty fruit bunches of oil palm. The size, shape, etc. of lignocellulosic biomass are not particularly limited, but from the viewpoint of improving the reaction efficiency in each step, those that have been pulverized and formed into chips are preferred.
前処理工程では、リグノセルロース系バイオマスの酵素糖化効率を高める処理を行う。前処理を行うことにより、セルロースおよびヘミセルロースへの酵素の接触が容易となり、酵素反応の効率が向上する。リグノセルロース系バイオマスの酵素糖化効率を高める処理としては、例えば、リグニンを可溶化する処理、セルロースおよびヘミセルロースとリグニンとの結合を切断する処理、セルロースおよびヘミセルロースの表面積を増大させる処理、セルロースの結晶性を低下させる処理などが挙げられる。本発明の糖化方法において前処理工程は必須ではなく、例えば古紙等のようにリグニン含量の低いリグノセルロース系バイオマスを用いる場合には省略することができる。前処理方法は、公知の方法から適宜選択することができる。例えば、アルカリ処理、酸処理、水熱処理、爆砕処理、有機溶媒処理、イオン性液体処理、マイクロ波処理、白色腐朽菌などのリグニン分解菌による処理、粉砕処理などが挙げられ、好ましくはアルカリ処理、酸処理、水熱処理であり、さらに好ましくはアルカリ処理である。 In the pretreatment step, a treatment for increasing the enzymatic saccharification efficiency of lignocellulosic biomass is performed. By performing the pretreatment, the enzyme is easily brought into contact with cellulose and hemicellulose, and the efficiency of the enzyme reaction is improved. Examples of the treatment for improving the enzymatic saccharification efficiency of lignocellulosic biomass include, for example, a treatment for solubilizing lignin, a treatment for cutting the bond between cellulose and hemicellulose and lignin, a treatment for increasing the surface area of cellulose and hemicellulose, and the crystallinity of cellulose. For example, a process for lowering. In the saccharification method of the present invention, the pretreatment step is not essential, and can be omitted when lignocellulosic biomass having a low lignin content such as waste paper is used. The pretreatment method can be appropriately selected from known methods. For example, alkali treatment, acid treatment, hydrothermal treatment, explosion treatment, organic solvent treatment, ionic liquid treatment, microwave treatment, treatment with lignin-degrading bacteria such as white rot fungus, pulverization treatment, etc., preferably alkali treatment, Acid treatment and hydrothermal treatment are preferred, and alkali treatment is more preferred.
前処理がアルカリ処理の場合、アルカリとしては、ナトリウム、カルシウム、カリウム、マグネシウムの水酸化物、酸化物、炭酸塩、炭酸水素塩、アンモニアなどが使用でき、好ましくは水酸化ナトリウム、炭酸ナトリウム、水酸化カルシウム、酸化カルシウム、アンモニアであり、さらに好ましくは水酸化ナトリウム、水酸化カルシウムである。アルカリは液状物（アルカリ液、好ましくはアルカリ水溶液）を用いてもよく、固状物を用いてもよい。固状のアルカリを用いる場合は、別途水を添加することが好ましい。アルカリ添加量は特に限定されないが、好ましくはリグノセルロース系バイオマスを含む原料に対して約１〜３０ｗｔ％、より好ましくは約５〜１５ｗｔ％添加する。なお、原料に対するｗｔ％は原料の乾燥重量（絶乾重量）に対するｗｔ％とし、以下も同様とする。処理温度は特に限定されないが、好ましくは約２０〜２００℃、より好ましくは約５０〜１５０℃である。処理時間は特に限定されないが、好ましくは約０．１〜１００時間、より好ましくは約１〜３０時間である。アルカリ処理後に、ろ過、遠心分離等により固液分離し、固形分を糖化工程に供する。分離後の固形分は、水等で洗浄してアルカリおよび可溶化リグニンを除去することが好ましい。また、洗浄後に乾燥させてもよいが、乾燥させずに糖化工程に供することが好ましい。また、リグニン分解を促進させるために、アントラキノン、スルホン化アントラキノンなどのアントラキノン類を添加してアルカリ処理を行ってもよい。 When the pretreatment is alkali treatment, sodium, calcium, potassium, magnesium hydroxide, oxide, carbonate, bicarbonate, ammonia, etc. can be used as the alkali, preferably sodium hydroxide, sodium carbonate, water Calcium oxide, calcium oxide, and ammonia are preferable, and sodium hydroxide and calcium hydroxide are more preferable. As the alkali, a liquid material (an alkali solution, preferably an aqueous alkali solution) may be used, or a solid material may be used. When using a solid alkali, it is preferable to add water separately. Although the amount of alkali added is not particularly limited, it is preferably added in an amount of about 1 to 30 wt%, more preferably about 5 to 15 wt% with respect to the raw material containing lignocellulosic biomass. The wt% with respect to the raw material is wt% with respect to the dry weight (absolute dry weight) of the raw material, and so on. Although processing temperature is not specifically limited, Preferably it is about 20-200 degreeC, More preferably, it is about 50-150 degreeC. The treatment time is not particularly limited, but is preferably about 0.1 to 100 hours, more preferably about 1 to 30 hours. After the alkali treatment, solid-liquid separation is performed by filtration, centrifugation, etc., and the solid content is subjected to a saccharification step. The solid content after the separation is preferably washed with water or the like to remove alkali and solubilized lignin. Moreover, although you may dry after washing | cleaning, it is preferable to use for a saccharification process, without making it dry. Moreover, in order to accelerate | stimulate lignin decomposition | disassembly, you may perform an alkali treatment by adding anthraquinones, such as anthraquinone and a sulfonated anthraquinone.
前処理が酸処理の場合は、硫酸、塩酸、硝酸、酢酸、リン酸等の酸類が使用でき、水溶液で用いるのが好ましく、希硫酸がさらに好ましい。酸添加量は特に限定されないが、好ましくはリグノセルロース系バイオマスを含む原料に対して約０．１〜１０ｗｔ％添加する。処理温度は特に限定されないが、好ましくは約１００〜２００℃である。処理時間は特に限定されないが、好ましくは約０．１〜１０時間である。固液分離、洗浄を行い、糖化工程に供する。 When the pretreatment is acid treatment, acids such as sulfuric acid, hydrochloric acid, nitric acid, acetic acid and phosphoric acid can be used, preferably in an aqueous solution, and more preferably dilute sulfuric acid. Although the acid addition amount is not particularly limited, it is preferably added in an amount of about 0.1 to 10 wt% with respect to the raw material containing lignocellulosic biomass. The treatment temperature is not particularly limited, but is preferably about 100 to 200 ° C. Although processing time is not specifically limited, Preferably it is about 0.1 to 10 hours. Solid-liquid separation and washing are performed, and the saccharification process is performed.
前処理が水熱処理の場合は、リグノセルロース系バイオマスを含む原料に水を添加し、高温高圧下で処理する。水の添加量は特に限定されないが、好ましくはリグノセルロース系バイオマスを含む原料に対して約１〜２０倍の重量を添加する。処理温度は特に限定されないが、好ましくは約１００〜２５０℃である。処理時間は特に限定されないが、好ましくは約０．１〜１０時間である。固液分離、洗浄を行ってもよいが、水熱処理の場合は、固液分離、洗浄を行わずに、糖化工程に供することもできる。 When the pretreatment is hydrothermal treatment, water is added to the raw material containing lignocellulosic biomass and the treatment is performed under high temperature and high pressure. The amount of water added is not particularly limited, but preferably about 1 to 20 times the weight of the raw material containing lignocellulosic biomass is added. The treatment temperature is not particularly limited, but is preferably about 100 to 250 ° C. Although processing time is not specifically limited, Preferably it is about 0.1 to 10 hours. Solid-liquid separation and washing may be performed, but in the case of hydrothermal treatment, the saccharification step may be performed without performing solid-liquid separation and washing.
これらの中でも、アルカリ処理が好ましい。後述するように、前処理工程におけるアルカリ処理により生じたアルカリ廃液を酵素回収工程のアルカリとして用いることにより、酵素回収の効率が向上することが確認されている（実施例７、実施例１０参照）。 Among these, alkali treatment is preferable. As will be described later, it has been confirmed that the efficiency of enzyme recovery is improved by using the alkaline waste liquid generated by the alkali treatment in the pretreatment process as an alkali in the enzyme recovery process (see Examples 7 and 10). .
糖化工程では、リグノセルロース系バイオマスを酵素で糖化する。糖化工程の原料には、上記前処理工程後のリグノセルロース系バイオマスを用いることが好ましい。用いる酵素は、セルロースを単糖（グルコース）に加水分解できる酵素、またはヘミセルロースを単糖（キシロース、マンノース、アラビノース等）に加水分解できる酵素を含むものであればよい。このような酵素は、一般にセルラーゼ、ヘミセルラーゼと称され、複数の酵素で構成される。本発明の糖化方法に用いる酵素は、セルラーゼまたはヘミセルラーゼを含むものであればよいが、糖化効率を向上させるために、両者の混合物を用いることが好ましい。セルラーゼとしては、セロビオヒドロラーゼ、β−グルカナーゼおよびβ−グルコシダーゼを含むものであることが好ましい。ヘミセルラーゼとしては、キシラナーゼおよびβ−キシロシダーゼを含むものであることが好ましい。他のヘミセルラーゼとしては、アセチルキシランエステラーゼ、α−アラビノフラノシダーゼ、マンナナーゼ、α−ガラクトシダーゼ、キシログルカナーゼ、ペクトリアーゼ、ペクチナーゼなどが挙げられる。また、植物細胞壁分解に関わる他の酵素、例えば、フェルラ酸エステラーゼ、クマル酸エステラーゼ、プロテアーゼなどを含んでいてもよい。これらの酵素を含有しているか否かは、各酵素の基質を用いて酵素活性を調べることにより、確認することができる。 In the saccharification step, lignocellulosic biomass is saccharified with an enzyme. It is preferable to use lignocellulosic biomass after the pretreatment step as a raw material for the saccharification step. The enzyme used may be an enzyme that can hydrolyze cellulose into a monosaccharide (glucose) or an enzyme that can hydrolyze hemicellulose into a monosaccharide (xylose, mannose, arabinose, etc.). Such an enzyme is generally called cellulase or hemicellulase, and is composed of a plurality of enzymes. Although the enzyme used for the saccharification method of this invention should just contain cellulase or hemicellulase, in order to improve saccharification efficiency, it is preferable to use the mixture of both. The cellulase preferably contains cellobiohydrolase, β-glucanase and β-glucosidase. The hemicellulase preferably contains xylanase and β-xylosidase. Other hemicellulases include acetyl xylan esterase, α-arabinofuranosidase, mannanase, α-galactosidase, xyloglucanase, pectinase, pectinase and the like. Further, it may contain other enzymes involved in plant cell wall degradation, such as ferulic acid esterase, coumaric acid esterase, and protease. Whether or not these enzymes are contained can be confirmed by examining the enzyme activity using the substrate of each enzyme.
酵素の由来としては特に限定されないが、トリコデルマ（Ｔｒｉｃｈｏｄｅｒｍａ）属、アクレモニウム属（Ａｃｒｅｍｏｎｉｕｍ）属、アスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属、ファネロケエテ（Ｐｈａｎｅｒｏｃｈａｅｔｅ）属、フーミコラ（Ｈｕｍｉｃｏｌａ）属、バチルス（Ｂａｃｉｌｌｕｓ）属の微生物などに由来する酵素が挙げられ、好ましくはトリコデルマ属、アクレモニウム属、アスペルギルス属由来の酵素であり、さらに好ましくはトリコデルマ属由来の酵素である。 The origin of the enzyme is not particularly limited, but includes the genus Trichoderma, the genus Acremonium, the genus Aspergillus, the genus Phanerocheete, the genus Humicola, and the genus Bacils. An enzyme derived from genus Trichoderma, Acremonium, and Aspergillus is preferable, and an enzyme derived from Trichoderma is more preferable.
これらの酵素は市販されており、本発明の糖化方法に好適に用いることができる。市販の酵素製剤としては、ノボザイムズ社製のＣｅｌｌｉｃシリーズ（ＣＴｅｃ２、ＨＴｅｃ２、ＣＴｅｃ、Ｈｔｅｃ）、ノボザイム１８８、Ｃｅｌｌｕｃｌａｓｔ、Ｐｕｌｐｚｙｍｅ、ジェネンコア社製のＡｃｃｅｌｌｅｒａｓｅシリーズ（Ｄｕｅｔ、１５００、ＸＹ、ＸＣ、ＢＧ）、Ｍｕｌｔｉｆｅｃｔシリーズ、明治製菓社製のメイセラーゼ、ヤクルト社製のオノズカ、アマノエンザイム社製のセルラーゼ（Ａ、Ｔ）などが挙げられる。好ましくはＣｅｌｌｉｃ ＣＴｅｃ２、Ｃｅｌｌｉｃ ＨＴｅｃ２、ＡｃｃｅｌｌｅｒａｓｅＤｕｅｔである。これらの酵素製剤はセロビオヒドロラーゼ、β−グルカナーゼ、β−グルコシダーゼ、キシラナーゼ、β−キシロシダーゼを含んでおり、原料のリグノセルロース系バイオマスの組成や含有酵素活性を考慮して、単独、あるいは複数を組み合わせて用いることができる。セルラーゼ活性の高い酵素製剤とヘミセルラーゼ活性の高い酵素製剤を組み合わせて用いる方法は本発明の好適な実施形態であり、特にＣｅｌｌｉｃ ＣＴｅｃ２とＣｅｌｌｉｃ ＨＴｅｃ２を組み合わせて混合して用いることが好ましい。 These enzymes are commercially available and can be suitably used for the saccharification method of the present invention. Examples of commercially available enzyme preparations include the Cellic series manufactured by Novozymes (CTec2, HTec2, CTec, Htec), Novozyme 188, Celluclast, Pulpzyme, Accelerase series (Duet 1500, XY, XC, BG) manufactured by Genencor Corporation, Multifect Meiji Seika's Mecellase, Yakult Onozuka, Amano Enzyme's Cellulase (A, T), and the like. Cellic CTec2, Cellic HTec2, and Accelerase Duet are preferable. These enzyme preparations contain cellobiohydrolase, β-glucanase, β-glucosidase, xylanase, and β-xylosidase. Considering the composition of the lignocellulosic biomass as a raw material and the contained enzyme activity, these enzyme preparations are used alone or in combination. Can be used. A method using a combination of an enzyme preparation having a high cellulase activity and an enzyme preparation having a high hemicellulase activity is a preferred embodiment of the present invention. In particular, it is preferable to use a combination of Cellic CTec2 and Cellic HTec2.
また本発明ではアルカリで酵素を回収して再利用することから、高いアルカリ安定性、および熱安定性を有する酵素を使用することが好ましい。酵素を化学的、あるいは遺伝子工学的（タンパク質工学的）に修飾してもよい。修飾することで、酵素安定性を高めたり、糖化反応残渣への吸着性を低減させたり、酵素回収効率を高めることができるため、本発明で好適に用いることができる。 In the present invention, an enzyme having high alkali stability and heat stability is preferably used because the enzyme is recovered and reused with an alkali. The enzyme may be modified chemically or genetically (protein engineering). By modifying, the enzyme stability can be increased, the adsorptivity to saccharification reaction residues can be reduced, and the enzyme recovery efficiency can be increased, so that it can be suitably used in the present invention.
糖化工程において、リグノセルロース系バイオマスを酵素処理して糖化する方法は特に限定されず、リグノセルロース系バイオマスを含む原料（以下「バイオマス原料」という。）と酵素が接触して単糖が生成される方法であればどのようなものでもよい。例えば、バイオマス原料に酵素および水を添加してスラリーを調製し、攪拌しながら反応させる方法が挙げられる。スラリーを攪拌せずに静置状態で反応を行ってもよいが、糖化反応促進のため攪拌することが好ましい。酵素の使用量としては特に限定されないが、好ましくはバイオマス原料に対して、酵素活性成分の重量として、約０．０１〜１０ｗｔ％、より好ましくは約０．０５〜３ｗｔ％添加する。水の添加量としては特に限定されないが、好ましくはバイオマス原料（乾燥重量）に対して約１〜２０倍、さらに好ましくは２〜１０倍量添加する。反応系には、酵素反応の妨げとならない限りバイオマス原料および酵素以外のものが添加されてもよい。例えば、テトラサイクリン塩酸塩、シクロヘキシミドなどの抗生物質を雑菌による糖消費の抑制の目的で添加してもよい。 In the saccharification step, the method of saccharifying lignocellulosic biomass by enzymatic treatment is not particularly limited, and a monosaccharide is produced by contact of a raw material containing lignocellulosic biomass (hereinafter referred to as “biomass raw material”) with the enzyme. Any method can be used. For example, a method may be mentioned in which an enzyme and water are added to a biomass raw material to prepare a slurry, which is reacted while stirring. The reaction may be carried out in a stationary state without stirring the slurry, but it is preferable to stir to promote the saccharification reaction. The amount of the enzyme used is not particularly limited, but is preferably about 0.01 to 10 wt%, more preferably about 0.05 to 3 wt%, based on the weight of the enzyme active component with respect to the biomass raw material. Although it does not specifically limit as addition amount of water, Preferably it is about 1-20 times with respect to biomass raw material (dry weight), More preferably, 2-10 times amount is added. As long as it does not interfere with the enzyme reaction, substances other than the biomass raw material and the enzyme may be added to the reaction system. For example, antibiotics such as tetracycline hydrochloride and cycloheximide may be added for the purpose of suppressing sugar consumption by various bacteria.
反応条件は、酵素による加水分解が進行する条件であれば特に限定されない。反応温度は通常約２０〜８０℃、好ましくは約３０〜６０℃である。反応時間は通常約１〜３００時間、好ましくは約１０〜１００時間である。反応ｐＨは酵素の至適ｐＨに従って設定すればよいが、通常ｐＨ約３〜７、好ましくはｐＨ約４．５〜６である。ｐＨコントロールのために、酢酸、クエン酸、コハク酸、リン酸などのバッファー成分を添加してもよい。ｐＨ調整には、酸、アルカリを適宜選択して用いることができる。一般に前処理工程としてアルカリ処理を行った場合には、バイオマス原料がアルカリ性になっているため、酸を使用して糖化に適したｐＨに調整する。この場合、使用する酸としては特に限定されないが、硫酸、塩酸、硝酸、リン酸、酢酸、クエン酸、コハク酸、二酸化炭素などが挙げられ、好ましくは硫酸、塩酸、酢酸、二酸化炭素である。また酵素のバイオマス原料への非特異的吸着を低減させるために、添加剤を添加してもよい。このような添加剤としては、タンパク質、界面活性剤、リグニン分解物などが挙げられ、好ましくは真菌や細菌などの微生物を培養して得られるタンパク質（菌体由来、および菌体外分泌タンパク質）、非イオン性界面活性剤、および脂肪酸由来の界面活性剤である。酵素の非特異的吸着を低減させることで、糖化速度の向上、酵素回収率の向上などが見込めるため、このような添加剤を添加することは本発明の好適な実施形態の一つである。 The reaction conditions are not particularly limited as long as the hydrolysis by the enzyme proceeds. The reaction temperature is usually about 20-80 ° C, preferably about 30-60 ° C. The reaction time is usually about 1 to 300 hours, preferably about 10 to 100 hours. The reaction pH may be set according to the optimum pH of the enzyme, but is usually about pH 3 to 7, preferably about pH 4.5 to 6. For pH control, buffer components such as acetic acid, citric acid, succinic acid and phosphoric acid may be added. For pH adjustment, an acid or an alkali can be appropriately selected and used. In general, when alkali treatment is performed as a pretreatment step, the biomass raw material is alkaline, so an acid is used to adjust to a pH suitable for saccharification. In this case, the acid to be used is not particularly limited, and examples thereof include sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, citric acid, succinic acid, carbon dioxide and the like, and sulfuric acid, hydrochloric acid, acetic acid and carbon dioxide are preferable. In addition, an additive may be added to reduce nonspecific adsorption of the enzyme to the biomass material. Examples of such additives include proteins, surfactants, lignin degradation products, and the like, preferably proteins obtained by culturing microorganisms such as fungi and bacteria (derived from cells and exocrine proteins), non- They are ionic surfactants and surfactants derived from fatty acids. By reducing non-specific adsorption of the enzyme, an improvement in saccharification rate, an improvement in enzyme recovery rate, and the like can be expected, so adding such an additive is one of the preferred embodiments of the present invention.
糖化工程により、酵素糖化反応の反応生成物である糖化反応スラリー（以下、単に「反応スラリー」という。）が得られる。この反応スラリーを、次の酵素回収工程に供する。反応スラリーは、糖化反応液（以下、単に「反応液」という。）と糖化反応残渣（以下、単に「反応残渣」という。）との混合物である。反応液には、グルコース、キシロース、マンノース、アラビノース等の単糖類および遊離の酵素が含まれている。反応残渣は反応液回収後の残りかすであり、未分解の糖質、リグニン、これらに吸着した酵素などが含まれている。 Through the saccharification step, a saccharification reaction slurry (hereinafter simply referred to as “reaction slurry”), which is a reaction product of the enzyme saccharification reaction, is obtained. This reaction slurry is subjected to the next enzyme recovery step. The reaction slurry is a mixture of a saccharification reaction liquid (hereinafter simply referred to as “reaction liquid”) and a saccharification reaction residue (hereinafter simply referred to as “reaction residue”). The reaction solution contains monosaccharides such as glucose, xylose, mannose, and arabinose and free enzymes. The reaction residue is a residual residue after the reaction solution is recovered, and contains undegraded carbohydrates, lignin, enzymes adsorbed to these, and the like.
酵素回収工程では、反応残渣に吸着した酵素を、アルカリを添加することで脱着させて回収する。本発明者らは、反応残渣と酵素との吸着／脱着状態は、系のｐＨに依存して変化することを見出した。すなわち、ｐＨを高くすると脱着率が高くなり（吸着率が低くなり）、ｐＨを低くすると吸着率が高くなる（脱着率が低くなる）ことを見出した。これは、反応残渣中のイオン性残基（カルボキシル基やフェノール性水酸基など）が、高ｐＨではイオン化されて親水化し、酵素／反応残渣の疏水的相互作用が弱まるためと考えられる。酵素の脱着率は、基本的には系のｐＨを高めるほど高くなるが、高いｐＨではアルカリによる酵素の失活が問題となる。そこで、本発明者らは、アルカリとの接触を制限しながら効率的に酵素回収を行うことが重要であると認識し、鋭意検討した結果、以下の（Ａ）および（Ｂ）の工程のうち少なくとも１つを含む酵素回収方法を見出した。
（Ａ）糖化反応スラリーにアルカリを添加して、糖化反応残渣に吸着した酵素を脱着させた後、糖化反応スラリーの固液分離を行って酵素を含有する酵素回収液を回収する工程
（Ｂ）糖化反応スラリーの固液分離により得られた糖化反応残渣にアルカリを添加して、糖化反応残渣に吸着した酵素を脱着させて酵素を含有する酵素回収液を回収し、その際、酵素回収液のｐＨが漸増するように糖化反応残渣にアルカリを添加する工程
本発明の糖化方法における酵素回収工程は、好ましくは工程（Ｂ）を含むものであり、より好ましくは工程（Ａ）および工程（Ｂ）を含むものである。
In the enzyme recovery step, the enzyme adsorbed on the reaction residue is desorbed and recovered by adding an alkali. The present inventors have found that the adsorption / desorption state between the reaction residue and the enzyme changes depending on the pH of the system. That is, it has been found that when the pH is increased, the desorption rate increases (adsorption rate decreases), and when the pH is decreased, the adsorption rate increases (desorption rate decreases). This is presumably because ionic residues (such as carboxyl groups and phenolic hydroxyl groups) in the reaction residue are ionized and hydrophilized at high pH, and the water / water interaction between the enzyme and the reaction residue is weakened. The desorption rate of the enzyme basically increases as the pH of the system increases, but at a high pH, the deactivation of the enzyme due to alkali becomes a problem. Therefore, the present inventors have recognized that it is important to efficiently recover the enzyme while restricting contact with alkali, and as a result of intensive studies, as a result of the following steps (A) and (B) We have found an enzyme recovery method comprising at least one.
(A) Step (B) of adding an alkali to the saccharification reaction slurry to desorb the enzyme adsorbed on the saccharification reaction residue and then performing solid-liquid separation of the saccharification reaction slurry to recover an enzyme recovery solution containing the enzyme. An alkali is added to the saccharification reaction residue obtained by solid-liquid separation of the saccharification reaction slurry, and the enzyme adsorbed on the saccharification reaction residue is desorbed to recover the enzyme recovery solution containing the enzyme. Step of adding alkali to saccharification reaction residue so that pH gradually increases The enzyme recovery step in the saccharification method of the present invention preferably includes step (B), more preferably step (A) and step (B). Is included.
背景技術で述べたリサイクル方法（ｃ）（非特許文献３、４、５参照、以下「方法（ｃ）」という。）は、本発明の酵素回収工程と同様にアルカリを用いて酵素回収を行うものである。しかしながら、方法（ｃ）は、固液分離後の反応残渣にアルカリを添加し酵素回収を行うのに対して、本発明の酵素回収工程における工程（Ａ）は、固液分離前の段階でアルカリを添加し酵素回収を行う点で異なっている。また、方法（ｃ）では、一定ｐＨのアルカリ液を用いて単一の酵素回収を行うのに対して、本発明の酵素回収工程における工程（Ｂ）は、低いｐＨから高いｐＨへ酵素回収液のｐＨを漸増させて酵素回収を行う点で異なっている。方法（ｃ）での酵素回収率は最大５０％程度にとどまっているが（ｐＨ１０付近）、これは、高いｐＨで単一のアルカリ処理をすることにより、酵素が失活しているためと考えられる。本発明の糖化方法における酵素回収工程は、よりマイルドな条件で酵素回収を行うものであるため、アルカリ失活することなく、高い酵素回収率を達成できるという点で優位である。 The recycling method (c) described in the background art (see Non-Patent Documents 3, 4, and 5; hereinafter referred to as “method (c)”) performs enzyme recovery using an alkali as in the enzyme recovery step of the present invention. Is. However, in the method (c), the alkali is added to the reaction residue after the solid-liquid separation and the enzyme is recovered, whereas the step (A) in the enzyme recovery step of the present invention is performed at the stage before the solid-liquid separation. In that the enzyme is recovered. In the method (c), a single enzyme is recovered using an alkaline solution having a constant pH, whereas the step (B) in the enzyme recovery step of the present invention is an enzyme recovery solution from a low pH to a high pH. The difference is that the pH is gradually increased to recover the enzyme. Although the enzyme recovery rate in the method (c) is only about 50% at maximum (around pH 10), it is considered that the enzyme is inactivated by a single alkali treatment at a high pH. It is done. The enzyme recovery step in the saccharification method of the present invention is advantageous in that a high enzyme recovery rate can be achieved without inactivating the alkali because the enzyme is recovered under milder conditions.
工程（Ａ）は、糖化工程で得られた反応スラリーに対して、まずアルカリを添加して反応スラリーのｐＨをアルカリ側にシフトさせ、反応残渣に吸着した酵素を一部脱着させて遊離の酵素を増加させる。続いて、ｐＨをシフトさせた反応スラリーの固液分離を行い、反応残渣を分離し、酵素回収液（＝反応液、単糖と遊離酵素を含む）を回収する。この操作により、遊離の酵素を増加させて単糖類と共に酵素回収液（＝反応液）中に回収できるため、より簡便に（酵素回収液を増加させずに）酵素回収率を高めることができる。すなわち、工程（Ａ）は、糖化工程後の反応液と反応残渣の固液分離の段階で酵素回収率を高めることができるという点が特徴であり、メリットである。酵素回収率をさらに高めたい場合は、分離した反応残渣からさらに酵素回収を行ってもよい。分離した反応残渣からさらに酵素を回収する方法は特に限定されないが、水洗浄、アルカリ添加が好ましく、さらに好ましくは後述する工程（Ｂ）の方法である。 In step (A), an alkali is first added to the reaction slurry obtained in the saccharification step, the pH of the reaction slurry is shifted to the alkali side, and a part of the enzyme adsorbed on the reaction residue is desorbed to release the enzyme. Increase. Subsequently, the reaction slurry having a shifted pH is subjected to solid-liquid separation, the reaction residue is separated, and the enzyme recovery solution (= reaction solution, containing monosaccharide and free enzyme) is recovered. By this operation, the free enzyme can be increased and recovered together with the monosaccharide in the enzyme recovery solution (= reaction solution), so that the enzyme recovery rate can be increased more easily (without increasing the enzyme recovery solution). That is, the step (A) is characterized in that the enzyme recovery rate can be increased at the stage of solid-liquid separation of the reaction solution and the reaction residue after the saccharification step, which is a merit. When it is desired to further increase the enzyme recovery rate, the enzyme may be further recovered from the separated reaction residue. The method of further recovering the enzyme from the separated reaction residue is not particularly limited, but water washing and alkali addition are preferred, and the method of step (B) described below is more preferred.
工程（Ａ）で反応スラリーに添加するアルカリとしては、アルカリ液が好ましく、アルカリ水溶液がより好ましい。アルカリ液またはアルカリ水溶液のｐＨは特に限定されないが、ｐＨが約７〜１４の範囲が好ましく、さらに好ましくはｐＨ約８〜１３の範囲である。アルカリとしては、ナトリウム、カルシウム、カリウム、マグネシウムの水酸化物、酸化物、炭酸塩、炭酸水素塩、アンモニアなどが使用でき、好ましくは水酸化ナトリウム、炭酸ナトリウム、水酸化カルシウム、酸化カルシウム、アンモニアであり、さらに好ましくは水酸化ナトリウム、水酸化カルシウムである。ホウ酸、リン酸などのバッファー成分を含んでいてもよい。またアルカリ液は酵素脱着を促進させるための添加剤を含んでいてもよい。このような添加剤としては、タンパク質、界面活性剤、リグニン分解物などが挙げられ、好ましくは真菌や細菌などの微生物を培養して得られるタンパク質（菌体由来、および菌体外分泌タンパク質）、非イオン性界面活性剤、および脂肪酸由来の界面活性剤である。 As the alkali added to the reaction slurry in the step (A), an alkali solution is preferable, and an aqueous alkali solution is more preferable. The pH of the alkaline solution or aqueous alkaline solution is not particularly limited, but the pH is preferably in the range of about 7 to 14, more preferably in the range of about pH 8 to 13. Sodium, calcium, potassium, magnesium hydroxide, oxide, carbonate, bicarbonate, ammonia, etc. can be used as the alkali, preferably sodium hydroxide, sodium carbonate, calcium hydroxide, calcium oxide, ammonia. More preferably sodium hydroxide or calcium hydroxide. Buffer components such as boric acid and phosphoric acid may be included. Moreover, the alkaline solution may contain an additive for promoting enzyme desorption. Examples of such additives include proteins, surfactants, lignin degradation products, and the like, preferably proteins obtained by culturing microorganisms such as fungi and bacteria (derived from cells and exocrine proteins), non- They are ionic surfactants and surfactants derived from fatty acids.
工程（Ａ）におけるアルカリ添加後の反応スラリーのｐＨは、中性〜弱アルカリ性であることが好ましい。これはアルカリによる酵素失活を抑制するためである。具体的には、ｐＨ約６〜１０であるのが好ましく、ｐＨ約６．５〜９であるのがさらに好ましい。添加するアルカリの量は、上記ｐＨを達成できる量であればよいが、アルカリ液として添加する場合は、反応スラリーの重量に対して約０．００１〜１００ｗｔ％の範囲が好ましく、約０．０１〜１０ｗｔ％の範囲がさらに好ましい。アルカリ添加後、反応残渣からの酵素脱着を促進させるために、攪拌、加熱等を行ってもよい。脱着時の温度は、約５〜６０℃の範囲が好ましく、約１０〜４０℃がさらに好ましい。脱着時間（アルカリ添加から固液分離までの時間）は、温度との兼合いで脱着率が最大化する時間を適宜設定すればよいが、一般的には約０．０１〜１０時間の範囲であり、約０．１〜３時間が好ましい。酵素脱着後の反応スラリーの固液分離方法は特に限定されず、例えば、ろ過、遠心分離、遠心ろ過、サイクロン、フィルタープレス、デカンターなどを使用することができる。工程（Ａ）においてアルカリ添加を行う反応スラリーは、糖化工程で得られる反応スラリーの全部でもよく、一部でもよい。工程（Ａ）における酵素回収は、回分式で行うことが好ましい。すなわち、アルカリ添加、脱着および固液分離は、段階的に行うことが好ましい。 The pH of the reaction slurry after the alkali addition in the step (A) is preferably neutral to weakly alkaline. This is to suppress enzyme deactivation by alkali. Specifically, the pH is preferably about 6 to 10, and more preferably about pH 6.5 to 9. The amount of alkali to be added may be any amount that can achieve the above pH, but when added as an alkali solution, the range of about 0.001 to 100 wt% is preferable with respect to the weight of the reaction slurry, about 0.01 The range of -10 wt% is more preferable. After the addition of alkali, stirring, heating, or the like may be performed in order to promote enzyme desorption from the reaction residue. The temperature at the time of desorption is preferably in the range of about 5 to 60 ° C, more preferably about 10 to 40 ° C. The desorption time (time from alkali addition to solid-liquid separation) may be set appropriately as long as the desorption rate is maximized in consideration of the temperature, but is generally in the range of about 0.01 to 10 hours. Yes, preferably about 0.1 to 3 hours. The solid-liquid separation method of the reaction slurry after enzyme desorption is not particularly limited, and for example, filtration, centrifugation, centrifugal filtration, cyclone, filter press, decanter and the like can be used. The reaction slurry to which alkali is added in the step (A) may be all or part of the reaction slurry obtained in the saccharification step. The enzyme recovery in the step (A) is preferably performed by a batch method. That is, the alkali addition, desorption, and solid-liquid separation are preferably performed in stages.
工程（Ｂ）は、まず糖化工程で得られた反応スラリーの固液分離を行い、反応液と反応残渣を分離する。続いて、得られた反応残渣に対して、アルカリを添加して酵素脱着を行い、酵素を含有する酵素回収液を回収する。工程（Ｂ）は、この反応残渣からの酵素回収において、回収された酵素を含む酵素回収液のｐＨが漸増するように反応残渣にアルカリを添加することを特徴する。ｐＨが漸増するとは、低いｐＨから高いｐＨへ徐々に変化することを意味する。この変化は連続的であっても、段階的であってもよいが、低いｐＨから酵素回収を開始し、ｐＨを増しながら酵素回収を行うことが重要である。セルラーゼおよびヘミセルラーゼはそれぞれ複数の酵素で構成されているが、個々の酵素が異なる反応残渣吸着特性および安定性（特にアルカリに対して）を有していることを本発明者らは確認している。ｐＨを漸増させて酵素回収を行うことで、アルカリとの接触を制限しながら、個々の酵素の特性に合ったｐＨ領域で酵素回収が行えるため、失活することなく高い酵素回収率を得ることができる。 In the step (B), first, solid-liquid separation of the reaction slurry obtained in the saccharification step is performed to separate the reaction solution and the reaction residue. Subsequently, an enzyme is desorbed by adding alkali to the obtained reaction residue, and an enzyme recovery solution containing the enzyme is recovered. The step (B) is characterized in that, in the enzyme recovery from the reaction residue, an alkali is added to the reaction residue so that the pH of the enzyme recovery solution containing the recovered enzyme gradually increases. Increasing pH means gradually changing from a low pH to a high pH. Although this change may be continuous or stepwise, it is important to start the enzyme recovery from a low pH and perform the enzyme recovery while increasing the pH. Although cellulase and hemicellulase are each composed of a plurality of enzymes, the present inventors have confirmed that each enzyme has different reaction residue adsorption characteristics and stability (especially against alkali). Yes. By performing enzyme recovery by gradually increasing the pH, enzyme recovery can be performed in a pH range that matches the characteristics of each enzyme while limiting contact with alkali, so that a high enzyme recovery rate can be obtained without inactivation. Can do.
工程（Ｂ）における反応スラリーの固液分離は、上記工程（Ａ）で述べた方法と同様の方法で行うことができる。反応スラリーとしては、糖化工程で得られた反応スラリーをそのまま用いてもよいが、工程（Ａ）によってアルカリ性に調整された反応スラリーを用いてもよい。反応残渣へのアルカリの添加は、反応残渣を適当な容器に入れ、アルカリを添加して反応残渣に吸着した酵素を脱着させる。容器は、糖化工程で用いた反応器をそのまま用いてもよい。添加するアルカリは、上記工程（Ａ）で述べたものと同様のものを用いることができ、添加剤も同様のものを用いることができる。アルカリと水とを別々に反応残渣に添加してもよいが、その場合は混合させて得られる溶液をアルカリ液として考える。また本発明者らは、塩濃度が高すぎると反応残渣からの酵素脱着が阻害されることを見出した。塩濃度は、アルカリ液中の塩濃度として、約０．０００１〜２００ｍＭであることが好ましく、さらに好ましくは約０．００１〜５０ｍＭである。添加するアルカリ液の総量としては、反応残渣（乾燥重量）に対して約１〜５００倍の範囲が好ましく、約３〜１００倍の範囲がさらに好ましい。アルカリ添加後、反応残渣からの酵素脱着を促進させるために、攪拌、加熱等を行ってもよい。脱着の条件は上記工程（Ａ）で述べた条件と同様の条件で行うことができる。続いて、ろ過、遠心分離等によりアルカリ脱着後の反応残渣を分離し、脱着した酵素を含む液を酵素回収液として回収する。回収した酵素回収液のｐＨが漸増していることは、酵素回収液を経時的に採取してｐＨを測定することで確認することができる。少なくともアルカリ添加後最初に採取した酵素回収液のｐＨより後に採取した酵素回収液のｐＨが高ければ、回収した酵素回収液のｐＨが漸増していると認められる。 The solid-liquid separation of the reaction slurry in the step (B) can be performed by a method similar to the method described in the step (A). As the reaction slurry, the reaction slurry obtained in the saccharification step may be used as it is, or the reaction slurry adjusted to be alkaline by the step (A) may be used. The alkali is added to the reaction residue by placing the reaction residue in a suitable container and adding the alkali to desorb the enzyme adsorbed on the reaction residue. As the container, the reactor used in the saccharification step may be used as it is. As the alkali to be added, the same ones as described in the above step (A) can be used, and the same additives can be used. Alkaline and water may be added separately to the reaction residue, but in that case, a solution obtained by mixing is considered as an alkaline solution. The inventors have also found that enzyme desorption from the reaction residue is inhibited when the salt concentration is too high. The salt concentration is preferably about 0.0001 to 200 mM, more preferably about 0.001 to 50 mM, as the salt concentration in the alkaline solution. The total amount of the alkali solution added is preferably about 1 to 500 times, more preferably about 3 to 100 times the reaction residue (dry weight). After the addition of alkali, stirring, heating, or the like may be performed in order to promote enzyme desorption from the reaction residue. The desorption conditions can be the same as the conditions described in the step (A). Subsequently, the reaction residue after alkali desorption is separated by filtration, centrifugation, etc., and a liquid containing the desorbed enzyme is recovered as an enzyme recovery liquid. It can be confirmed that the pH of the collected enzyme recovery solution gradually increases by collecting the enzyme recovery solution over time and measuring the pH. It is recognized that the pH of the collected enzyme recovery solution is gradually increased if the pH of the enzyme recovery solution collected at least after the pH of the enzyme recovery solution collected first after the alkali addition is high.
工程（Ｂ）での反応残渣からの酵素回収は、連続式で行ってもよく、回分式で行ってもよい。連続式の方が、より少ないアルカリ液の量で効率的に酵素回収を行えるため、好ましい。工程（Ｂ）を連続式で行う場合は、アルカリ液の添加および脱着した酵素を含む酵素回収液の回収を、いずれも連続的に行うことができる。連続的に添加するアルカリ液の組成、ｐＨは、添加開始から添加終了まで一定でもよく、変化してもよい。変化する場合は、添加開始時のｐＨより添加終了時のｐＨが高いことが好ましい。アルカリ液のｐＨの変化は連続的であってもよく、段階的であってもよい。酵素回収液のｐＨが漸増していることは、例えば、回収開始時、回収途中、回収終了時の酵素回収液を適宜採取してｐＨを測定することで確認することができる。測定した酵素回収液のｐＨが、回収開始時＜回収途中＜回収終了時となっていればよい。 The enzyme recovery from the reaction residue in the step (B) may be carried out continuously or batchwise. The continuous type is preferable because the enzyme can be efficiently recovered with a smaller amount of alkaline solution. In the case where the step (B) is performed continuously, the addition of the alkaline solution and the recovery of the enzyme recovery solution containing the desorbed enzyme can be performed continuously. The composition and pH of the alkali solution to be continuously added may be constant from the start of addition to the end of addition or may be changed. When changing, the pH at the end of the addition is preferably higher than the pH at the start of the addition. The pH change of the alkaline solution may be continuous or stepwise. The gradual increase in the pH of the enzyme recovery liquid can be confirmed, for example, by appropriately collecting the enzyme recovery liquid at the start of recovery, during recovery, or at the end of recovery and measuring the pH. It is only necessary that the measured pH of the enzyme recovery solution is such that the recovery start time <recovery in progress <recovery end time.
工程（Ｂ）を回分式で行う場合は、反応残渣へのアルカリ液の添加および脱着した酵素を含む酵素回収液の回収を少なくとも２回以上繰り返す。１回目に添加するアルカリ液の組成、ｐＨと、２回目以後に添加するアルカリ液の組成、ｐＨは同じでもよく、異なってもよい。１回目と２回目以後に異なるｐＨのアルカリ液を添加する場合は、後の回のアルカリ液のｐＨが前の回のアルカリ液のｐＨより高いことが好ましい。酵素回収液のｐＨが漸増していることは、各回の酵素回収液のｐＨを測定することにより確認することができる。後の回の酵素回収液のｐＨが、前の回の酵素回収液のｐＨより高くなっていればよい。 When the step (B) is performed batchwise, the addition of the alkaline solution to the reaction residue and the recovery of the enzyme recovery solution containing the desorbed enzyme are repeated at least twice. The composition and pH of the alkaline solution added at the first time and the composition and pH of the alkaline solution added after the second time may be the same or different. When adding alkaline solutions having different pHs after the first time and after the second time, it is preferable that the pH of the subsequent alkaline solution is higher than the pH of the previous alkaline solution. The gradual increase in the pH of the enzyme recovery solution can be confirmed by measuring the pH of the enzyme recovery solution each time. The pH of the subsequent enzyme recovery solution only needs to be higher than the pH of the previous enzyme recovery solution.
工程（Ｂ）において、反応残渣にアルカリを添加後、ｐＨ漸増の終点における酵素回収液のｐＨは、約７〜１３であることが好ましく、約７．５〜１１であることがより好ましい。当該ｐＨ範囲で酵素回収を終了することで、十分に高い酵素回収率を得ることができる。「ｐＨ漸増の終点」とは、酵素回収液のｐＨを漸増させて酵素回収を行う区間の終点を意味する。通常、酵素回収区間の全範囲が酵素回収液のｐＨを漸増させて酵素回収を行う区間に該当するので、ｐＨ漸増の終点における酵素回収液は酵素回収終了時の酵素回収液を意味することになる。すなわち、通常ｐＨ漸増の終点における酵素回収液は、連続式で酵素回収液を回収する場合には、酵素回収終了時の回収液を含み、ｐＨ測定が可能となる一定量（通常約１０〜１００ｍＬ）の酵素回収液を意味し、回分式で酵素回収液を回収する場合には、最終回の酵素回収液を意味する。ただし、酵素回収工程の途中または終了時にｐＨを低下させる操作を行う場合には、ｐＨを低下させる操作を行う前に存在する酵素回収液のｐＨ漸増区間の終点が、「ｐＨ漸増の終点」になる。
酵素回収工程における工程（Ｂ）の特徴は、反応残渣からの酵素回収において酵素回収液のｐＨを漸増させることであるので、酵素回収開始時の酵素回収液のｐＨは、酵素回収終了時よりも低いｐＨであればよく、特に限定されない。反応残渣からの酵素回収開始時の酵素回収液のｐＨは、ｐＨ約４〜１１であることが好ましく、ｐＨ約５〜９であることがより好ましい。また、酵素回収工程において、アルカリ添加による酵素回収区間の途中または終了後に、水などを用いて反応残渣を洗浄し、残存する酵素を回収してもよい。
In step (B), after adding alkali to the reaction residue, the pH of the enzyme recovery solution at the end of the gradual increase in pH is preferably about 7 to 13, and more preferably about 7.5 to 11. A sufficiently high enzyme recovery rate can be obtained by terminating the enzyme recovery within the pH range. The “end point of gradual increase in pH” means the end point of a section in which enzyme recovery is performed by gradually increasing the pH of the enzyme recovery solution. In general, the entire range of the enzyme recovery section corresponds to a section where the enzyme recovery solution is gradually increased in pH to perform enzyme recovery, so that the enzyme recovery solution at the end of the gradual increase in pH means the enzyme recovery solution at the end of enzyme recovery. Become. That is, the enzyme recovery solution at the end point of normal pH gradual increase, when recovering the enzyme recovery solution in a continuous manner, includes a recovery solution at the end of enzyme recovery, and a certain amount (usually about 10 to 100 mL) that enables pH measurement. In the case of collecting the enzyme recovery solution in batch mode, it means the last enzyme recovery solution. However, when performing an operation for lowering the pH during or at the end of the enzyme recovery step, the end point of the pH gradually increasing section of the enzyme recovery solution existing before the operation for lowering the pH is the `` pH gradually increasing end point ''. Become.
Since the characteristic of the step (B) in the enzyme recovery step is to gradually increase the pH of the enzyme recovery solution in the enzyme recovery from the reaction residue, the pH of the enzyme recovery solution at the start of enzyme recovery is higher than that at the end of enzyme recovery. There is no particular limitation as long as the pH is low. The pH of the enzyme recovery solution at the start of enzyme recovery from the reaction residue is preferably about pH 4-11, more preferably about pH 5-9. Further, in the enzyme recovery step, the reaction residue may be washed with water or the like to recover the remaining enzyme during or after the enzyme recovery section by addition of alkali.
リグノセルロース系バイオマスから単糖への糖化効率を高めるために、糖化工程で用いる酵素として複数のセルラーゼおよび複数のヘミセルラーゼの混合物を用いることが好ましいが、個々の酵素が糖化反応残渣から脱着し易いｐＨはそれぞれ異なることが確認されている。例えば、セルラーゼ活性を有するβ−グルコシダーゼはｐＨが１０を超えるような領域のほうが脱着しやすく、同じくセルラーゼ活性を有するセロビオヒドロラーゼはより低いｐＨの領域でも脱着することを、本発明者らは確認している（実施例２〜５参照）。また一方、アルカリ安定性に関しては、β−グルコシダーゼは高く、セロビオヒドロラーゼは低いという違いがあることも、本発明者らは確認している。酵素回収工程における工程（Ｂ）は、酵素回収液のｐＨが漸増するように反応残渣にアルカリを添加するものであるため、脱着容易なｐＨおよび安定性が異なる複数の酵素混合物（例えば、複数のセルラーゼおよび複数のヘミセルラーゼの混合物）を糖化酵素として使用した場合でも高い酵素回収率を実現することができる点で、非常に有用である。 In order to increase the saccharification efficiency from lignocellulosic biomass to monosaccharides, it is preferable to use a mixture of a plurality of cellulases and a plurality of hemicellulases as an enzyme used in the saccharification process, but each enzyme is easily desorbed from a saccharification reaction residue. It has been confirmed that the pH is different. For example, the present inventors have confirmed that β-glucosidase having cellulase activity is more easily desorbed in a region where the pH exceeds 10 and cellobiohydrolase having cellulase activity is also desorbed in a lower pH region. (See Examples 2 to 5). On the other hand, regarding the alkali stability, the present inventors have also confirmed that there is a difference that β-glucosidase is high and cellobiohydrolase is low. In step (B) in the enzyme recovery step, an alkali is added to the reaction residue so that the pH of the enzyme recovery solution gradually increases. Therefore, a plurality of enzyme mixtures (for example, a plurality of enzyme mixtures different in pH and stability that are easy to desorb) are used. This is very useful in that a high enzyme recovery rate can be realized even when a cellulase and a mixture of hemicellulases are used as saccharifying enzymes.
前処理工程がアルカリ処理の場合、前処理工程においてリグノセルロース系バイオマスをアルカリ処理して生じたアルカリ廃液を、酵素回収工程における工程（Ａ）および／または工程（Ｂ）のアルカリとして用いることが好ましい。本発明者らは、前処理工程で生じたアルカリ廃液を、酵素回収工程のアルカリ液に対して１０ｗｔ％添加して酵素回収を行うことにより、アルカリ廃液を添加していないアルカリ液で酵素回収を行う場合より酵素回収効率が向上すること（実施例６参照）を見出した。また、前処理工程で生じたアルカリ廃液を、酵素回収工程のアルカリ液に対して５ｗｔ％添加して酵素回収を行うことにより、アルカリ廃液を添加していないアルカリ液で酵素回収を行う場合より少ない処理回数で同等の酵素回収率が得られること（実施例１０）を見出した。このアルカリ廃液の添加効果は、廃液中のリグニン水溶化物などの疎水性化合物によって、酵素の反応残渣からの脱着が促進されるため、回収効率が向上すると考えられる。酵素回収工程のアルカリとして、前処理工程で生じたアルカリ廃液のみ（アルカリ廃液１００ｗｔ％）を用いてもよいが、前処理工程で生じたアルカリ廃液の添加量は、酵素回収工程のアルカリ液に対して約５０ｗｔ％以下が好ましく、約２５ｗｔ％以下がより好ましい。さらに好ましくは、酵素回収工程のアルカリ液に対して約１〜１０ｗｔ％である。 In the case where the pretreatment step is an alkali treatment, it is preferable to use the alkali waste liquid produced by alkali treatment of lignocellulosic biomass in the pretreatment step as the alkali in the step (A) and / or step (B) in the enzyme recovery step. . The present inventors perform enzyme recovery by adding 10 wt% of the alkaline waste liquid generated in the pretreatment process to the alkaline liquid in the enzyme recovery process, thereby recovering the enzyme with the alkaline liquid to which the alkaline waste liquid has not been added. It has been found that the enzyme recovery efficiency is improved as compared with the case of carrying out (see Example 6). Moreover, the amount of the alkaline waste liquid generated in the pretreatment process is 5 wt% with respect to the alkaline liquid in the enzyme recovery process, and the enzyme recovery is performed, thereby reducing the amount of the enzyme recovered in the alkaline liquid without adding the alkaline waste liquid. It was found that the same enzyme recovery rate was obtained by the number of treatments (Example 10). The effect of adding the alkaline waste liquid is thought to improve the recovery efficiency because the desorption of the enzyme from the reaction residue is promoted by a hydrophobic compound such as a lignin aqueous solution in the waste liquid. As the alkali in the enzyme recovery process, only the alkali waste liquid generated in the pretreatment process (alkaline waste liquid 100 wt%) may be used. However, the amount of the alkali waste liquid generated in the pretreatment process is larger than that in the enzyme recovery process. About 50 wt% or less is preferable, and about 25 wt% or less is more preferable. More preferably, it is about 1-10 wt% with respect to the alkaline solution of an enzyme collection process.
工程（Ａ）および／または工程（Ｂ）で得た酵素回収液は、回収後速やかに酸を添加してｐＨを弱酸性〜中性に調整することが好ましい。酸を添加後の酵素回収液のｐＨとしては、ｐＨ４〜７であるのが好ましく、ｐＨ４．５〜６であるのがさらに好ましい。酵素回収液は回収後、速やかに酸を添加することが好ましいが、酸を添加するまでの時間としては、０〜２４時間であることがより好ましく、０〜１０時間であることがさらに好ましい。このような方法で酵素回収液を管理することで、アルカリによる酵素失活を防ぐことができる。添加する酸としては、例えば、硫酸、塩酸、硝酸、酢酸、クエン酸、コハク酸、リン酸などが挙げられる。 It is preferable that the enzyme recovery solution obtained in the step (A) and / or the step (B) is adjusted to have a slightly acidic to neutral pH by adding an acid immediately after the recovery. The pH of the enzyme recovery solution after addition of the acid is preferably pH 4-7, more preferably pH 4.5-6. The enzyme recovery solution is preferably added with acid immediately after recovery, but the time until the acid is added is preferably 0 to 24 hours, more preferably 0 to 10 hours. By managing the enzyme recovery solution by such a method, enzyme inactivation due to alkali can be prevented. Examples of the acid to be added include sulfuric acid, hydrochloric acid, nitric acid, acetic acid, citric acid, succinic acid, and phosphoric acid.
得られた酵素回収液は、糖化工程に再利用することができる。必要に応じて、酵素回収液を限外ろ過などの方法で濃縮してから再利用する。また反応スラリーを固液分離して得られた反応液も遊離の酵素（および単糖類）を含むため、限外ろ過などの方法で酵素と単糖類とを分離して酵素を再利用することが好ましい。またバイオマス原料への吸着現象を利用して、酵素を簡便に再利用することもできる。すなわち、単糖類などを含む反応液、もしくは酵素回収液をフレッシュのバイオマス原料と接触させる。バイオマス原料には酵素のみが吸着するため、固液分離によって単糖類と酵素（バイオマス原料に吸着した状態）を分離することができる。また、単糖類を発酵原料として用いる場合には、単糖類を含む酵素回収液をそのまま発酵に供してもよい。この場合、得られる発酵液から菌体および発酵生産物を除去した後、発酵液残渣（酵素を含む）を糖化工程に利用することで、簡便に酵素の再利用を行うことができる。酵素回収液を糖化反応に再利用する際は、フレッシュな酵素を一部追加してもよい。追加する酵素は、初回に使用した酵素組成と同様でもよいが、回収酵素は酵素構成が変化している場合があるので、回収酵素の活性に合わせて、適宜追加の酵素を選択することが好ましい。例えば、β−グルコシダーゼは反応残渣に吸着しやすく、他の酵素に比べて回収率が低くなる場合があるため、そのような場合はβ−グルコシダーゼを多く含む酵素液を追加することが好ましい。 The obtained enzyme recovery solution can be reused in the saccharification step. If necessary, the enzyme recovery solution is concentrated by a method such as ultrafiltration and reused. In addition, since the reaction liquid obtained by solid-liquid separation of the reaction slurry also contains free enzyme (and monosaccharide), the enzyme and monosaccharide can be separated and reused by methods such as ultrafiltration. preferable. Further, the enzyme can be easily reused by utilizing the adsorption phenomenon to the biomass raw material. That is, a reaction solution containing a monosaccharide or the like or an enzyme recovery solution is brought into contact with a fresh biomass raw material. Since only the enzyme is adsorbed to the biomass material, the monosaccharide and the enzyme (the state adsorbed to the biomass material) can be separated by solid-liquid separation. Moreover, when using monosaccharide as a fermentation raw material, you may use for the fermentation the enzyme recovery liquid containing a monosaccharide as it is. In this case, after removing the microbial cells and the fermentation product from the obtained fermentation broth, the enzyme can be easily reused by using the fermentation broth residue (including the enzyme) in the saccharification step. When reusing the enzyme recovery solution for the saccharification reaction, a part of fresh enzyme may be added. The enzyme to be added may be the same as the enzyme composition used for the first time. However, since the enzyme composition of the recovered enzyme may have changed, it is preferable to select an additional enzyme as appropriate in accordance with the activity of the recovered enzyme. . For example, β-glucosidase is likely to be adsorbed on the reaction residue and the recovery rate may be lower than that of other enzymes. In such a case, it is preferable to add an enzyme solution containing a large amount of β-glucosidase.
得られた単糖類の用途は特に限定されないが、発酵原料、化学品原料、飼料、肥料などに好適に用いることができる。発酵原料として用いる場合には、エタノール、１−ブタノール、イソブタノール、２−プロパノール、乳酸、コハク酸、酢酸、３−ヒドロキシプロピオン酸、ピルビン酸、クエン酸、１，３−プロパンジオールなどの化学品の発酵生産に好適に用いることができる。 Although the use of the obtained monosaccharide is not particularly limited, it can be suitably used for fermentation raw materials, chemical raw materials, feeds, fertilizers and the like. When used as a fermentation raw material, chemicals such as ethanol, 1-butanol, isobutanol, 2-propanol, lactic acid, succinic acid, acetic acid, 3-hydroxypropionic acid, pyruvic acid, citric acid, 1,3-propanediol It can use suitably for fermentation production of.
各工程で用いる装置は特に限定されないが、前処理工程、および糖化工程で用いる反応器は、例えばバッチ式、連続式、半連続式の装置などを用いることができる。具体的には、フィルターを備えたバッチ式反応槽、スクリューフィーダー式の連続反応器、原料添加と反応液抜き出しを連続的に行う半連続式反応槽、カラム式の充填反応槽などが挙げられる。固液分離装置としては、フィルタープレス、遠心分離、遠心ろ過、サイクロン、デカンターなどを用いることができる。酵素回収工程では、糖化工程と同様の装置（反応器）を用いることができるが、好ましくはアルカリ液の添加と酵素回収液の抜き出しを連続的に行うことができる装置である。糖化工程の装置をそのまま用いてもよい。 Although the apparatus used in each process is not particularly limited, the reactor used in the pretreatment process and the saccharification process may be, for example, a batch type, continuous type, or semi-continuous type apparatus. Specific examples include a batch-type reaction tank equipped with a filter, a screw feeder-type continuous reactor, a semi-continuous reaction tank for continuously adding raw materials and extracting a reaction liquid, and a column-type packed reaction tank. A filter press, centrifugal separation, centrifugal filtration, cyclone, decanter, etc. can be used as the solid-liquid separation device. In the enzyme recovery step, the same apparatus (reactor) as in the saccharification step can be used, but preferably an apparatus capable of continuously adding an alkaline solution and extracting the enzyme recovery solution. You may use the apparatus of a saccharification process as it is.
本発明の糖化方法を用いて回収される酵素の回収率（糖化工程で使用した酵素の酵素活性に対する回収された酵素の酵素活性）は非常に高いので、回収された酵素を有効にリサイクル使用することができる。本発明の糖化方法において、反応残渣から回収される酵素と、反応液から回収される酵素を合わせると、糖化工程で使用した酵素量に対して、酵素活性として少なくとも約５０％以上、条件により約７０％以上が回収可能である。したがって、本発明の糖化方法は、リグノセルロース系バイオマスの糖化に用いる酵素の使用量を減らし、酵素コストを大幅に低減することができる点で、非常に有用な技術である。 Since the recovery rate of the enzyme recovered using the saccharification method of the present invention (the enzyme activity of the recovered enzyme relative to the enzyme activity of the enzyme used in the saccharification step) is very high, the recovered enzyme is effectively recycled. be able to. In the saccharification method of the present invention, when the enzyme recovered from the reaction residue and the enzyme recovered from the reaction solution are combined, the enzyme activity is at least about 50% or more with respect to the amount of enzyme used in the saccharification step. More than 70% can be recovered. Therefore, the saccharification method of the present invention is a very useful technique in that the amount of enzyme used for saccharification of lignocellulosic biomass can be reduced and the enzyme cost can be greatly reduced.
以下、実施例により本発明を詳細に説明するが、本発明はこれらに限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.
〔実験材料〕
（１）バイオマス
リグノセルロース系バイオマスとして、パーム油を生産する際に排出されるアブラヤシの空果房（以下「ＥＦＢ」という。）を原料に用いた（産地インドネシア）。
（２）糖化酵素
ノボザイムズ社の酵素液Ｃｅｌｌｉｃ ＣＴｅｃ２（以下「ＣＴｅｃ」という。）およびＣｅｌｌｉｃ ＨＴｅｃ２（以下「ＨＴｅｃ」という。）を用いた。これらの酵素液は、セルラーゼとしてセロビオヒドロラーゼ（以下「ＣＢＨ」という。）、β−グルコシダーゼ（以下「ＧＬＤ」という。）、β−グルカナーゼ（以下「ＣＭＣ」という。）を含有し、ヘミセルラーゼとしてキシラナーゼ（以下「ＸＹＮ」という。）、キシロシダーゼ（以下「ＸＬＤ」という。）を含有している。
[Experimental material]
(1) Biomass As lignocellulosic biomass, oil palm empty fruit bunches (hereinafter referred to as “EFB”) discharged when producing palm oil were used as raw materials (production area Indonesia).
(2) Saccharifying Enzyme The enzyme solutions Cellic CTec2 (hereinafter referred to as “CTec”) and Cellic HTec2 (hereinafter referred to as “HTec”) manufactured by Novozymes were used. These enzyme solutions contain cellobiohydrolase (hereinafter referred to as “CBH”), β-glucosidase (hereinafter referred to as “GLD”), and β-glucanase (hereinafter referred to as “CMC”) as cellulases, and as hemicellulases. It contains xylanase (hereinafter referred to as “XYN”) and xylosidase (hereinafter referred to as “XLD”).
〔分析方法〕
酵素活性の測定、および酵素回収率の算出は、以下のようにして行った。なお、ここで使用したバッファーは０．１Ｍ、ｐＨ５．０の酢酸バッファーである。
（１）ＣＢＨ活性
ｐ−ニトロフェニル−β−Ｄ−セロビオシド（ＰＮＰ−ＣＢ）を基質とした比色法により測定した。すなわち、１．５ｍｌのマイクロチューブに２１５μｌのバッファーをとり、酵素含有のサンプル溶液を１０μｌ加えた。これに基質溶液（ＰＮＰ−ＣＢの１．２５ｗｔ％バッファー溶液）を２５μｌ加えて酵素反応を開始し、水浴上で４０℃、１時間保温して酵素反応を行った。１時間経過後、０．１Ｍのグリシン水溶液（ｐＨ１０．０）を５００μｌ添加し、酵素反応の停止と発色を行った。得られたサンプルの吸光度（波長４０５ｎｍ）を測定し、酵素活性の指標とした。測定は、吸光度が１．５を越えない範囲で、適宜バッファーで希釈したサンプル溶液を用いて行った。酵素回収液、および糖化反応に使用したものと同じ酵素液（初期投入酵素液）の酵素活性を測定し、以下の式に従って回収酵素活性（＝酵素回収率）を算出した。
回収酵素活性（％）＝［酵素回収液中の酵素活性総量／初期投入酵素液中の酵素活性総量］×１００
（２）ＧＬＤ活性
ｐ−ニトロフェニル−β−Ｄ−グルコピラノシド（ＰＮＰ−ＧＰ）を基質とした比色法により測定した。ＣＢＨ活性と全く同様の方法で、ただし基質をＰＮＰ−ＧＰ、酵素反応条件を４０℃、３０分に変え、測定を行った。
[Analysis method]
Measurement of enzyme activity and calculation of enzyme recovery were performed as follows. In addition, the buffer used here is an acetic acid buffer of 0.1 M and pH 5.0.
(1) CBH activity The CBH activity was measured by a colorimetric method using p-nitrophenyl-β-D-cellobioside (PNP-CB) as a substrate. That is, 215 μl of buffer was taken in a 1.5 ml microtube, and 10 μl of enzyme-containing sample solution was added. 25 μl of a substrate solution (PNP-CB 1.25 wt% buffer solution) was added thereto to start the enzyme reaction, and the enzyme reaction was carried out by keeping it on a water bath at 40 ° C. for 1 hour. After 1 hour, 500 μl of 0.1 M aqueous glycine solution (pH 10.0) was added to stop the enzyme reaction and develop color. The absorbance (wavelength 405 nm) of the obtained sample was measured and used as an index of enzyme activity. The measurement was performed using a sample solution appropriately diluted with a buffer as long as the absorbance does not exceed 1.5. The enzyme activity of the enzyme recovery solution and the same enzyme solution (initially charged enzyme solution) used for the saccharification reaction was measured, and the recovered enzyme activity (= enzyme recovery rate) was calculated according to the following formula.
Recovery enzyme activity (%) = [total enzyme activity in enzyme recovery solution / total enzyme activity in initial input enzyme solution] × 100
(2) GLD activity It was measured by a colorimetric method using p-nitrophenyl-β-D-glucopyranoside (PNP-GP) as a substrate. Measurement was carried out in the same manner as CBH activity except that the substrate was PNP-GP and the enzyme reaction conditions were changed to 40 ° C. for 30 minutes.
（３）ＣＭＣ活性（β−グルカナーゼ）
カルボキシメチルセルロース（ＣＭＣ）を基質とした比色法により測定した。還元糖量の測定にはＤＮＳ（ジニトロサリチル酸）法を用いた。すなわち、１．５ｍｌのマイクロチューブに１８０μｌの基質溶液（カルボキシメチルセルロースナトリウム塩、シグマ社製Ｃ５６７８の４ｗｔ％バッファー溶液）をとり、酵素含有のサンプル溶液を２０μｌ加えて酵素反応を開始し、水浴上で５０℃、１時間保温して酵素反応を行った。１時間経過後、３００μｌのＤＮＳ溶液Ａ（１％３，５−ジニトロサリチル酸、０．２％フェノール、１％水酸化ナトリウム、０．０５％亜硫酸ナトリウムの水溶液）および１００μｌのＤＮＳ溶液Ｂ（４０％ロッシェル塩水溶液）を加え、沸騰水中で１０分間加熱して発色させ、水冷後、６００μｌの水を加えて希釈した。得られたサンプルの吸光度（波長５４０ｎｍ）を測定した（吸光度１）。サンプル中の初期含有糖量を差し引くために、酵素反応を行わないサンプル（酵素サンプルをＤＮＳ溶液の後に加える）を同様に調製し、発色を行って吸光度を測定した（吸光度２）。吸光度１と吸光度２の差を酵素活性の指標とした。測定は各吸光度が１．５を越えない範囲で、適宜バッファーで希釈したサンプル溶液を用いて行った。回収酵素活性はＣＢＨ活性と同様に算出した。
(3) CMC activity (β-glucanase)
It was measured by a colorimetric method using carboxymethylcellulose (CMC) as a substrate. A DNS (dinitrosalicylic acid) method was used to measure the amount of reducing sugar. Specifically, 180 μl of a substrate solution (carboxymethylcellulose sodium salt, 4 wt% buffer solution of C5678 made by Sigma) was taken into a 1.5 ml microtube, and 20 μl of an enzyme-containing sample solution was added to start the enzyme reaction. The enzyme reaction was performed by incubating at 50 ° C. for 1 hour. After 1 hour, 300 μl DNS solution A (1% 3,5-dinitrosalicylic acid, 0.2% phenol, 1% sodium hydroxide, 0.05% sodium sulfite aqueous solution) and 100 μl DNS solution B (40% Rochelle salt aqueous solution) was added, and the mixture was heated for 10 minutes in boiling water to cause color development. After cooling with water, 600 μl of water was added for dilution. The absorbance (wavelength 540 nm) of the obtained sample was measured (absorbance 1). In order to subtract the initial sugar content in the sample, a sample in which no enzyme reaction was carried out (the enzyme sample was added after the DNS solution) was prepared in the same manner, color development was performed, and the absorbance was measured (absorbance 2). The difference between absorbance 1 and absorbance 2 was used as an index of enzyme activity. The measurement was performed using a sample solution appropriately diluted with a buffer as long as each absorbance does not exceed 1.5. The recovered enzyme activity was calculated in the same manner as the CBH activity.
（４）ＸＹＮ活性
可溶性キシランを基質とした比色法により測定した。ＣＭＣ活性と全く同様の方法で、ただし基質を可溶性キシラン（２ｗｔ％キシラン、シグマ社製Ｘ０５０２、のバッファー溶液を調製し、不溶分を遠心分離で取り除いた上清画分を使用）、および酵素反応条件を４０℃、３０分に変え、測定を行った。
（５）ＸＬＤ活性
ｐ−ニトロフェニル−β−Ｄ−キシロピラノシド（ＰＮＰ−ＸＰ）を基質とした比色法により測定した。ＣＢＨ活性と全く同様の方法で、ただし基質をＰＮＰ−ＸＰに変え、測定を行った。
(4) XYN activity It was measured by a colorimetric method using soluble xylan as a substrate. In exactly the same manner as the CMC activity, except that the substrate is soluble xylan (2 wt% xylan, Sigma X0502 buffer solution prepared and the insoluble content removed by centrifugation is used) and enzyme reaction The conditions were changed to 40 ° C. and 30 minutes, and measurement was performed.
(5) XLD activity Measured by a colorimetric method using p-nitrophenyl-β-D-xylopyranoside (PNP-XP) as a substrate. Measurement was performed in the same manner as CBH activity except that the substrate was changed to PNP-XP.
糖化反応における生成糖類の定量分析は、ＨＰＬＣ（高速液体クロマトグラフィー）を使用して行った。カラムは東ソー社ＴＳＫｇｅｌ Ａｍｉｄｅ−８０を用い、示差屈折計（ＲＩ）にて検出を行った。移動相としてはアセトニトリル／水＝７５／２５（体積比）を用い、カラム温度は８０℃にて分析を行った。
糖類の収率は重量基準で算出した。すなわち、以下の式で算出した。
糖収率（ｗｔ％）＝［生成糖の重量／原料バイオマスの乾燥重量］×１００
Quantitative analysis of the produced saccharide in the saccharification reaction was performed using HPLC (high performance liquid chromatography). The column was detected with a differential refractometer (RI) using TSKgel Amide-80 manufactured by Tosoh Corporation. As the mobile phase, acetonitrile / water = 75/25 (volume ratio) was used, and the column temperature was analyzed at 80 ° C.
The yield of saccharides was calculated on a weight basis. That is, it was calculated by the following formula.
Sugar yield (wt%) = [weight of produced sugar / dry weight of raw material biomass] × 100
〔実施例１〕
（１）前処理工程（アルカリ前処理、１０ｗｔ％ＮａＯＨ）
１００ｍｌの高圧反応器に、粉砕処理（１ｍｍスクリーン）を施した原料ＥＦＢ（６．０ｇ、含水率７．３％）、水酸化ナトリウム（０．６ｇ＝１０ｗｔ％対原料ＥＦＢ）、および水（２９．４ｇ）を加えて密閉した。反応器を１２０℃のオイルバスで３時間加熱し、冷却後、ろ過により固液分離を行い、さらに固形分を水洗浄した。得られた固形分を１０５℃で３時間乾燥し、前処理ＥＦＢ−１（４．１ｇ、含水率８．７％、絶乾基準の重量収率６７．３％）を取得した。
[Example 1]
(1) Pretreatment step (alkali pretreatment, 10 wt% NaOH)
Raw material EFB (6.0 g, water content 7.3%), sodium hydroxide (0.6 g = 10 wt% to raw material EFB), and water (29) subjected to pulverization (1 mm screen) in a 100 ml high-pressure reactor 4 g) was added and sealed. The reactor was heated in an oil bath at 120 ° C. for 3 hours, cooled, solid-liquid separation was performed by filtration, and the solid content was washed with water. The obtained solid content was dried at 105 ° C. for 3 hours to obtain pretreated EFB-1 (4.1 g, water content 8.7%, weight yield 67.3% based on absolute dryness).
（２）糖化工程
１０ｍｌのガラス容器に、前処理ＥＦＢ−１（０．４４ｇ）を加え、さらに酵素溶液（ＣＴｅｃ３ｍｇ／ｍｌ、ＨＴｅｃ３ｍｇ／ｍｌ、酢酸バッファー（ｐＨ５．５）０．０５Ｍ、テトラサイクリン塩酸塩４０μｇ／ｍｌ、シクロヘキシミド３０μｇ／ｍｌを含有）を６ｍｌ加え、密閉した。これを恒温振とう機で振とうしながら、４５℃で２４時間、糖化反応を行い、反応スラリーを得た。
(2) Saccharification step Pretreatment EFB-1 (0.44 g) is added to a 10 ml glass container, and further enzyme solution (CTec 3 mg / ml, HTec 3 mg / ml, acetate buffer (pH 5.5) 0.05 M, tetracycline hydrochloride 6 ml) (containing 40 μg / ml and cycloheximide 30 μg / ml) was added and sealed. While shaking this with a constant temperature shaker, a saccharification reaction was carried out at 45 ° C. for 24 hours to obtain a reaction slurry.
（３）酵素回収工程（工程（Ｂ））
得られた反応スラリーを遠心ろ過カラム（モビテック社のモビコール、フィルター細孔径３０μｍ）に入れ、遠心分離にかけ、反応液と反応残渣に固液分離した（以下、各実施例の番号を付して「反応液１」、「反応残渣１」等という。）。反応液１を分析したところ、グルコース収率は４３．１ｗｔ％、キシロース収率は１５．５ｗｔ％で合計５８．６ｗｔ％の単糖類の生成が確認された（前処理ＥＦＢ基準）。なお未処理の原料ＥＦＢ基準に換算すると、グルコース収率は２９．０ｗｔ％、キシロース収率は１０．４ｗｔ％で合計３９．４ｗｔ％となる。
得られた反応残渣１にｐＨ８．０のアルカリ液（０．０５Ｍホウ酸ナトリウムバッファー）を５ｍｌ添加し、室温で３０分間攪拌混合して吸着酵素の脱着処理を行った。遠心ろ過を行い、酵素回収液１−１（ｐＨ７．１）と残渣に分離した。次にｐＨ９．０のアルカリ液（０．０５Ｍホウ酸ナトリウムバッファー）５ｍｌを用いて同様に脱着処理を行い、酵素回収液１−２（ｐＨ８．８）と残渣を得た。続いてｐＨ１０．０のアルカリ液（０．０５Ｍホウ酸ナトリウムバッファー）５ｍｌを用いて同様に脱着処理を行い、酵素回収液１−３（ｐＨ９．８）と残渣を得た。なお酵素回収液には速やかに酢酸バッファーを添加し、ｐＨを５〜７の範囲に調整して保存した。
(3) Enzyme recovery process (process (B))
The obtained reaction slurry was put into a centrifugal filtration column (Mobitech's Mobicol, filter pore size 30 μm), and centrifuged to separate the reaction solution and the reaction residue into solid and liquid (hereinafter, the numbers of the respective examples are attached to “ Reaction liquid 1 ”,“ reaction residue 1 ”, etc.). When the reaction solution 1 was analyzed, it was confirmed that the glucose yield was 43.1 wt%, the xylose yield was 15.5 wt%, and a total of 58.6 wt% monosaccharides were produced (based on pretreatment EFB). In terms of the raw material EFB standard, the glucose yield is 29.0 wt% and the xylose yield is 10.4 wt%, which is 39.4 wt% in total.
5 ml of pH 8.0 alkaline solution (0.05 M sodium borate buffer) was added to the obtained reaction residue 1, and the adsorbed enzyme was desorbed by stirring and mixing at room temperature for 30 minutes. Centrifugal filtration was performed to separate the enzyme recovery liquid 1-1 (pH 7.1) and the residue. Next, a desorption treatment was similarly performed using 5 ml of pH 9.0 alkaline solution (0.05 M sodium borate buffer) to obtain enzyme recovery solution 1-2 (pH 8.8) and a residue. Subsequently, desorption treatment was similarly performed using 5 ml of an alkaline solution (0.05 M sodium borate buffer) at pH 10.0 to obtain an enzyme recovery solution 1-3 (pH 9.8) and a residue. The enzyme recovery solution was quickly added with an acetate buffer, adjusted to a pH of 5 to 7, and stored.
反応液１、酵素回収液１−１〜３に含まれる５種の酵素活性（ＣＢＨ、ＧＬＤ、ＣＭＣ、ＸＹＮ、ＸＬＤ）を測定し、回収酵素活性（初期投入量に対する％で表示）を表１に示した。表１の結果から、アルカリ液のｐＨを徐々に上げてアルカリ処理することで酵素回収液のｐＨを徐々に上げることにより、高い酵素回収率が得られることが分かった。特に、吸着率の高いＧＬＤも高い割合で回収可能であることが分かった。 Five enzyme activities (CBH, GLD, CMC, XYN, XLD) contained in Reaction Solution 1 and Enzyme Recovery Solution 1-1 to 1-3 were measured, and the recovered enzyme activity (expressed as a percentage of the initial input amount) is shown in Table 1. It was shown to. From the results of Table 1, it was found that a high enzyme recovery rate can be obtained by gradually increasing the pH of the enzyme recovery solution by gradually increasing the pH of the alkaline solution and performing an alkali treatment. In particular, it was found that GLD having a high adsorption rate can be recovered at a high rate.
（４）酵素再利用工程
反応液１、酵素回収液１−１〜３を混合し、限外ろ過を行った。すなわち、遠心分離式の限外ろ過装置（クラボウ社のセントリカット、限外ろ過膜の材質はポリスルホン、分画分子量１万）を用い、回収酵素液を濃縮した。さらに酢酸バッファー（０．０５Ｍ、ｐＨ５．５）で洗浄を行い、最終的に酵素液を約４ｍｌに調整し、酵素回収液１−４を得た。続いて、上記初回の糖化工程と同様の方法で前処理ＥＦＢ−１の糖化反応を行った。ただし、酵素溶液としては、酵素回収液１−４に初回の２０％分のフレッシュ酵素（ＣＴｅｃとＨＴｅＣの比率は１対１）を補充したものを使用した。４５℃、２４時間の糖化反応で、グルコース収率は４３．０ｗｔ％、キシロース収率は１３．５ｗｔ％、合計５６．５ｗｔ％（前処理ＥＦＢ基準）であり、１００％フレッシュ酵素を用いた初回の反応と同等の糖収率が得られた。すなわち、酵素使用量の８０％が削減できた。
(4) Enzyme recycling step Reaction solution 1 and enzyme recovery solutions 1-1 to 1-3 were mixed and subjected to ultrafiltration. That is, the recovered enzyme solution was concentrated using a centrifugal ultrafiltration apparatus (Cenbow cut by Kurabo Industries, the material of the ultrafiltration membrane is polysulfone, molecular weight cut off 10,000). Furthermore, it wash | cleaned with the acetate buffer (0.05M, pH5.5), finally adjusted the enzyme liquid to about 4 ml, and obtained the enzyme collection | recovery liquid 1-4. Subsequently, a saccharification reaction of pretreated EFB-1 was performed in the same manner as in the first saccharification step. However, as the enzyme solution, the enzyme recovery solution 1-4 supplemented with the initial 20% fresh enzyme (the ratio of CTec to HTeC was 1: 1) was used. In the saccharification reaction at 45 ° C. for 24 hours, the glucose yield was 43.0 wt%, the xylose yield was 13.5 wt%, the total was 56.5 wt% (based on pretreatment EFB), and the first time using 100% fresh enzyme A sugar yield equivalent to that in the above reaction was obtained. That is, 80% of the enzyme usage was reduced.
〔実施例２〕
（１）前処理工程、糖化工程
実施例１と同様に前処理工程を行い、ＥＦＢ前処理品を得た。糖化工程においては、酵素濃度をＣＴｅｃ４．８ｍｇ／ｍｌ、ＨＴｅｃ１．２ｍｇ／ｍｌ（割合にして８対２）に変更し、反応温度および時間を５０℃、７２時間に変更した以外は実施例１と同様に行い、反応スラリーを得た。
[Example 2]
(1) Pretreatment step and saccharification step A pretreatment step was carried out in the same manner as in Example 1 to obtain an EFB pretreatment product. In the saccharification step, Example 1 was used except that the enzyme concentration was changed to CTec 4.8 mg / ml, HTec 1.2 mg / ml (ratio 8 to 2), and the reaction temperature and time were changed to 50 ° C. and 72 hours. The reaction slurry was obtained in the same manner.
（２）酵素回収工程（工程（Ｂ））
続いて、実施例１と同様に得られた反応スラリーを遠心ろ過にかけ、反応液２と反応残渣２に分離した。反応液２を分析したところ、グルコース収率は４６．８ｗｔ％、キシロース収率は２３．３ｗｔ％で合計７０．１ｗｔ％の単糖類の生成が確認された（前処理ＥＦＢ基準）。得られた反応残渣２にｐＨ９．０のアルカリ液（０．１Ｍホウ酸ナトリウムバッファー）を５ｍｌ添加し、室温で３０分間攪拌混合して吸着酵素の脱着処理を行った。遠心分離を行い、上澄みの酵素回収液（ｐＨ８．４）と残渣に分離した。再度、ｐＨ９．０のアルカリ液（０．１Ｍホウ酸ナトリウムバッファー）５ｍｌを用いて同様に脱着処理を行い、酵素回収液（ｐＨ８．８）と残渣を得た。再び、ｐＨ９．０のアルカリ液（０．１Ｍホウ酸ナトリウムバッファー）５ｍｌを用いて同様に脱着処理を行い、酵素回収液（ｐＨ９．０）と残渣を得た。得られた３つの酵素回収液は混合し、酵素回収液２とした。
(2) Enzyme recovery process (process (B))
Subsequently, the reaction slurry obtained in the same manner as in Example 1 was subjected to centrifugal filtration to separate into reaction solution 2 and reaction residue 2. When the reaction solution 2 was analyzed, it was confirmed that the glucose yield was 46.8 wt%, the xylose yield was 23.3 wt%, and a total of 70.1 wt% monosaccharides were generated (based on pretreatment EFB). 5 ml of a pH 9.0 alkaline solution (0.1 M sodium borate buffer) was added to the obtained reaction residue 2, and the adsorbed enzyme was desorbed by stirring and mixing at room temperature for 30 minutes. Centrifugation was performed to separate the supernatant into an enzyme recovery solution (pH 8.4) and a residue. Again, a desorption treatment was performed in the same manner using 5 ml of an alkaline solution (pH 0.1 M sodium borate buffer) to obtain an enzyme recovery solution (pH 8.8) and a residue. Again, a desorption treatment was performed in the same manner using 5 ml of pH 9.0 alkaline solution (0.1 M sodium borate buffer) to obtain an enzyme recovery solution (pH 9.0) and a residue. The obtained three enzyme recovery solutions were mixed to obtain enzyme recovery solution 2.
反応液２および酵素回収液２のＧＬＤ活性およびＣＢＨ活性を測定した。ＧＬＤは、反応液２中に５％、酵素回収液２中に５１％の回収酵素活性が確認された。ＣＢＨは、反応液２中に６０％、酵素回収液２中に２２％の回収酵素活性が確認された。アルカリ処理のｐＨパターン（酵素回収液のｐＨ）と共に表２に回収酵素活性をまとめた。ｐＨが同じアルカリ液を用いて脱着処理を３回繰り返した場合、酵素回収液のｐＨは段階的に上がり、高い酵素回収率が得られることが分かった。 The GLD activity and CBH activity of reaction solution 2 and enzyme recovery solution 2 were measured. As for GLD, the recovered enzyme activity was confirmed to be 5% in the reaction solution 2 and 51% in the enzyme recovery solution 2. The recovered enzyme activity of CBH was confirmed to be 60% in the reaction solution 2 and 22% in the enzyme recovery solution 2. The recovered enzyme activity is summarized in Table 2 together with the pH pattern of the alkali treatment (pH of the enzyme recovery solution). It was found that when the desorption treatment was repeated three times using an alkaline solution having the same pH, the pH of the enzyme recovery solution increased stepwise, and a high enzyme recovery rate was obtained.
〔実施例３〜５〕
実施例２と同様に前処理、糖化、酵素回収工程（工程（Ｂ））を実施した。ただし酵素回収工程では、アルカリ液として、実施例３ではｐＨ９．５、実施例４ではｐＨ１０．０、実施例５ではｐＨ１０．５の０．１Ｍホウ酸ナトリウムバッファーを用いて、それぞれ３回の脱着処理を行い酵素回収液３〜５を得た。酵素回収液中のＧＬＤ活性およびＣＢＨ活性を測定し、得られたＧＬＤおよびＣＢＨの回収酵素活性と、３回のアルカリ処理のｐＨパターンを表２に示した。
実施例２〜５の結果から、ＧＬＤに関しては高いｐＨの方がより高い回収率が得られ、ＣＢＨに関しては低いｐＨの方がより高い回収率が得られることが分かった。このように、酵素種によって脱着しやすいｐＨ条件が異なることが分かった。
[Examples 3 to 5]
In the same manner as in Example 2, pretreatment, saccharification, and enzyme recovery step (step (B)) were performed. However, in the enzyme recovery step, desorption was performed 3 times each using 0.1 M sodium borate buffer at pH 9.5 in Example 3, pH 10.0 in Example 4, and pH 10.5 in Example 5 as an alkaline solution. It processed and obtained enzyme recovery liquids 3-5. The GLD activity and CBH activity in the enzyme recovery solution were measured, and the recovered enzyme activity of the obtained GLD and CBH and the pH pattern of the three alkali treatments are shown in Table 2.
From the results of Examples 2 to 5, it was found that a higher recovery rate was obtained with a high pH for GLD, and a higher recovery rate was obtained with a low pH for CBH. Thus, it was found that the pH conditions at which desorption is easy depend on the enzyme species.
〔実施例６〕
実施例２と同様に前処理、糖化、酵素回収工程（工程（Ｂ））を実施した。ただし酵素回収工程では、アルカリ液として、前処理工程で得られたアルカリ廃液（固液分離後のろ液原液）を０．１Ｍホウ酸バッファーに対して１０ｗｔ％添加した溶液（ｐＨ９．０）を用い、３回の脱着処理（アルカリ処理のｐＨパターンは実施例２と同じ）を行って酵素回収液６を得た。ＧＬＤ活性を測定したところ、酵素回収液６中に５８％の活性が確認された。同ｐＨ条件の実施例２と比較して（５１％）、回収率の向上が観察され、前処理液に酵素脱着促進効果が有ることが分かった。
Example 6
In the same manner as in Example 2, pretreatment, saccharification, and enzyme recovery step (step (B)) were performed. However, in the enzyme recovery step, a solution (pH 9.0) obtained by adding 10 wt% of the alkaline waste solution (filtrate stock solution after solid-liquid separation) obtained in the pretreatment step to the 0.1 M borate buffer is used as the alkaline solution. The enzyme recovery solution 6 was obtained by performing desorption treatment three times (the pH pattern of the alkali treatment was the same as in Example 2). When the GLD activity was measured, 58% activity was confirmed in the enzyme recovery solution 6. Compared to Example 2 under the same pH condition (51%), an improvement in the recovery rate was observed, and it was found that the pretreatment liquid had an enzyme desorption promoting effect.
〔実施例７〕
（１）前処理工程（アルカリ前処理、７．５ｗｔ％ＮａＯＨ）
１００ｍｌの高圧反応器に、実施例１で用いた原料ＥＦＢ粉砕品を１．００ｇ、水酸化ナトリウムを７５ｍｇ（＝７．５ｗｔ％対原料ＥＦＢ）、および水１０．０ｇを加えて密閉した。反応器を１５０℃のオイルバスで３時間加熱し、冷却後、ろ過により固液分離を行い、さらに固形分を水洗浄して前処理ＥＦＢ−２（水ウェット体）を得た。得られた固形分は乾燥工程を経ずに次の糖化工程に供した。
Example 7
(1) Pretreatment step (alkali pretreatment, 7.5 wt% NaOH)
To a 100 ml high-pressure reactor, 1.00 g of the raw EFB pulverized product used in Example 1, 75 mg of sodium hydroxide (= 7.5 wt% vs. raw EFB), and 10.0 g of water were added and sealed. The reactor was heated in an oil bath at 150 ° C. for 3 hours, cooled, solid-liquid separation was performed by filtration, and the solid content was washed with water to obtain a pretreated EFB-2 (water wet body). The obtained solid content was subjected to the next saccharification step without passing through the drying step.
（２）糖化工程
２０ｍｌのガラス容器に、前処理ＥＦＢ−２を全量、ＣＴｅｃを３０ｍｇ、ＨＴｅｃを３０ｍｇ、０．１Ｍ酢酸バッファー（ｐＨ５．５）を５ｍｌ、テトラサイクリン塩酸塩を４００μｇ、シクロヘキシミドを３００μｇ加えた。１０％酢酸水溶液でｐＨを５．５に調整した後、最後に反応液の重量を水で１１．０ｇに調整した。これを振とうしながら、４５℃で４４時間糖化反応を行い、反応スラリーを得た。
(2) Saccharification step In a 20 ml glass container, add the entire amount of pretreated EFB-2, 30 mg of CTec, 30 mg of HTec, 5 ml of 0.1 M acetate buffer (pH 5.5), 400 μg of tetracycline hydrochloride, and 300 μg of cycloheximide. It was. After adjusting the pH to 5.5 with a 10% aqueous acetic acid solution, the weight of the reaction solution was finally adjusted to 11.0 g with water. While shaking this, a saccharification reaction was carried out at 45 ° C. for 44 hours to obtain a reaction slurry.
（３）酵素回収工程（工程（Ｂ））
続いて、実施例１と同様に得られた反応スラリーを遠心ろ過にかけ、反応液７と反応残渣７に分離した。反応液７を分析したところ、グルコース収率は３２．７ｗｔ％、キシロース収率は１２．７ｗｔ％で合計４５．４ｗｔ％の単糖類の生成が確認された（未処理の原料ＥＦＢ基準）。得られた反応残渣７に水を５ｍｌ加え、攪拌混合してスラリーとした。続いて、スラリーのｐＨを測定しながら、０．１％のＮａＯＨ水溶液（ｐＨ１２．４）を適量添加し、スラリーｐＨを６．６に調整した。これを３０℃で３０分間振とうし、アルカリ液による酵素脱着操作を行った。続いて遠心ろ過を行い、酵素回収液７−１（ｐＨ６．６）と残渣を得た。残渣に対して更に同様の操作を２回繰り返し（表３に示したｐＨに調整）、酵素回収液７−２（ｐＨ７．１）および酵素回収液７−３（ｐＨ８．２）を得た。本実施例では、０．１％ＮａＯＨ水溶液を用いて、処理液のｐＨを段階的に上げる方法で脱着操作を行った。
(3) Enzyme recovery process (process (B))
Subsequently, the reaction slurry obtained in the same manner as in Example 1 was subjected to centrifugal filtration to separate into reaction solution 7 and reaction residue 7. When the reaction solution 7 was analyzed, it was confirmed that the glucose yield was 32.7 wt%, the xylose yield was 12.7 wt%, and a total of 45.4 wt% monosaccharides were produced (untreated raw material EFB standard). 5 ml of water was added to the obtained reaction residue 7 and stirred to form a slurry. Subsequently, while measuring the pH of the slurry, an appropriate amount of 0.1% NaOH aqueous solution (pH 12.4) was added to adjust the slurry pH to 6.6. This was shaken at 30 ° C. for 30 minutes, and an enzyme desorption operation with an alkaline solution was performed. Subsequently, centrifugal filtration was performed to obtain an enzyme recovery solution 7-1 (pH 6.6) and a residue. The same operation was further repeated twice on the residue (adjusted to the pH shown in Table 3) to obtain enzyme recovery solution 7-2 (pH 7.1) and enzyme recovery solution 7-3 (pH 8.2). In this example, the desorption operation was performed by using a 0.1% NaOH aqueous solution in a stepwise manner to raise the pH of the treatment liquid.
反応液７、酵素回収液７−１〜３に含まれる５種の酵素活性を測定し、回収酵素活性（初期投入量に対する％で表示）を表３に示した。 Five types of enzyme activities contained in the reaction solution 7 and the enzyme recovery solutions 7-1 to 3 were measured, and the recovered enzyme activities (expressed in% relative to the initial input amount) are shown in Table 3.
〔実施例８〕
実施例７と同様に、前処理、糖化、酵素回収工程（工程（Ｂ））を行った。ただし酵素回収工程では、アルカリ処理ｐＨを表４に示したｐＨパターンに変えて、３回の脱着操作を行った。得られた反応液８、回収酵素液８−１〜３に含まれる５種の酵素活性を測定し、回収酵素活性（初期投入量に対する％で表示）を表４に示した。
Example 8
In the same manner as in Example 7, pretreatment, saccharification, and enzyme recovery step (step (B)) were performed. However, in the enzyme recovery process, the alkali treatment pH was changed to the pH pattern shown in Table 4, and the desorption operation was performed three times. Five kinds of enzyme activities contained in the obtained reaction solution 8 and the collected enzyme solutions 8-1 to 3 were measured, and the collected enzyme activities (expressed in% with respect to the initial input amount) are shown in Table 4.
〔実施例９〕
実施例７と同様に、前処理、糖化、酵素回収工程（工程（Ｂ））を行った。ただし酵素回収工程では、０．１％ＮａＯＨの代わりに、０．１％Ｃａ（ＯＨ）２水溶液を用いて、ｐＨの調整を行った。また、アルカリ処理ｐＨは、表５に示したｐＨパターンで３回の脱着操作を行った。得られた反応液９、回収酵素液９−１〜３に含まれる５種の酵素活性を測定し、回収酵素活性（初期投入量に対する％で表示）を表５に示した。
Example 9
In the same manner as in Example 7, pretreatment, saccharification, and enzyme recovery step (step (B)) were performed. However, in the enzyme recovery step, pH was adjusted using a 0.1% Ca (OH) 2 aqueous solution instead of 0.1% NaOH. Further, the alkali treatment pH was carried out three times with the pH pattern shown in Table 5. Five types of enzyme activities contained in the obtained reaction solution 9 and the collected enzyme solutions 9-1 to 3 were measured, and the collected enzyme activities (expressed in% relative to the initial input amount) are shown in Table 5.
〔実施例１０〕
実施例７と同様に、前処理、糖化、酵素回収工程（工程（Ｂ））を行った。ただし酵素回収工程では、アルカリ液として、前処理工程で得られたアルカリ廃液（固液分離後のろ液原液）を０．１％のＮａＯＨ水溶液に対して５ｗｔ％添加した溶液（ｐＨ１２．４）を用いた。また脱着操作は３回ではなく、２回に変更した。得られた反応液１０、回収酵素液１０−１、２に含まれる５種の酵素活性を測定し、回収酵素活性（初期投入量に対する％で表示）を表６に示した。この結果から、前処理液を添加することで、２回の脱着操作で３回の脱着操作（実施例７）と同等の回収率が得られることが分かった。
Example 10
In the same manner as in Example 7, pretreatment, saccharification, and enzyme recovery step (step (B)) were performed. However, in the enzyme recovery step, a solution (pH 12.4) obtained by adding 5 wt% of the alkaline waste solution (filtrate after solid-liquid separation) obtained in the pretreatment step to the 0.1% NaOH aqueous solution as the alkaline solution. Was used. The desorption operation was changed to 2 times instead of 3 times. Five enzyme activities contained in the obtained reaction solution 10 and the recovered enzyme solutions 10-1 and 10-2 were measured, and the recovered enzyme activities (expressed in% relative to the initial input amount) are shown in Table 6. From this result, it was found that by adding the pretreatment liquid, a recovery rate equivalent to three desorption operations (Example 7) can be obtained by two desorption operations.
〔実施例１１〕酵素回収工程：工程（Ａ）
実施例７と同様に、前処理、糖化工程を行った。得られた反応スラリー（ｐＨ５．５）に１％水酸化ナトリウム水溶液を適量加え、ｐＨを７．９に上げて、吸着酵素の一部脱着を行った。続いて、実施例７と同様に遠心ろ過で固液分離を行い、酵素回収液１１−１と反応残渣１１を得た。反応残渣１１に水を５ｍｌ加え、攪拌混合して３０℃で３０分間振とうし、水による洗浄操作を行った。遠心ろ過を行い、酵素回収液１１−２（ｐＨ８．２）と残渣を得た。
[Example 11] Enzyme recovery step: Step (A)
In the same manner as in Example 7, a pretreatment and a saccharification step were performed. An appropriate amount of 1% aqueous sodium hydroxide solution was added to the resulting reaction slurry (pH 5.5) to raise the pH to 7.9, and partial desorption of the adsorbed enzyme was performed. Subsequently, solid-liquid separation was performed by centrifugal filtration in the same manner as in Example 7 to obtain enzyme recovery solution 11-1 and reaction residue 11. 5 ml of water was added to the reaction residue 11, stirred and mixed, shaken at 30 ° C. for 30 minutes, and washed with water. Centrifugal filtration was performed to obtain an enzyme recovery solution 11-2 (pH 8.2) and a residue.
酵素回収液１１−１〜２に含まれる５種の酵素活性を測定し、回収酵素活性を表７に示した。実施例７と比較して、反応液（酵素回収液１１−１）中に含まれる酵素活性が増加しており、反応スラリーのｐＨを上げることで遊離の酵素量を増やすことができ、より効率的に酵素回収を行うことができることが分かった。 Five enzyme activities contained in the enzyme recovery solutions 11-1 and 2 were measured, and the recovered enzyme activities are shown in Table 7. Compared with Example 7, the enzyme activity contained in the reaction solution (enzyme recovery solution 11-1) is increased, and the amount of free enzyme can be increased by raising the pH of the reaction slurry, which is more efficient. It was found that enzyme recovery can be carried out automatically.
〔実施例１２〕
（１）前処理工程（アルカリ前処理、１０ｗｔ％ＮａＯＨ、アントラキノン添加）
実施例７と同様に行った。ただしここでは、加えるＮａＯＨの量を１００ｍｇ（＝１０ｗｔ％対原料ＥＦＢ）に変更し、さらに助剤としてアントラキノン−２−スルホン酸ナトリウム１水和物（東京化成）を１０ｍｇ加え、加熱条件を１２０℃、３時間にして実験を行い、前処理ＥＦＢ−３を得た。得られた前処理品は乾燥工程を経ずに次の糖化工程に供した。
Example 12
(1) Pretreatment step (alkali pretreatment, addition of 10 wt% NaOH and anthraquinone)
The same operation as in Example 7 was performed. However, here, the amount of NaOH to be added is changed to 100 mg (= 10 wt% vs. raw EFB), and 10 mg of sodium anthraquinone-2-sulfonate monohydrate (Tokyo Kasei) is added as an auxiliary agent, and the heating condition is 120 ° C. The experiment was conducted for 3 hours to obtain pre-treated EFB-3. The obtained pretreated product was subjected to the next saccharification step without passing through the drying step.
（２）糖化工程
実施例７と同様に行った。ただしここでは、糖化反応時間を４８時間に変更して実験を行い、反応スラリー１１．０ｇを得た。反応スラリーのｐＨは５．６であった。反応スラリーから反応液を少量抜き出し（反応液１２とする）、分析したところ、グルコース収率は３３．５ｗｔ％、キシロース収率は１４．９ｗｔ％で合計４８．３ｗｔ％の単糖類の生成が確認された（未処理の原料ＥＦＢ基準）。
(2) Saccharification process It carried out similarly to Example 7. However, here, the experiment was conducted by changing the saccharification reaction time to 48 hours to obtain 11.0 g of a reaction slurry. The pH of the reaction slurry was 5.6. When a small amount of the reaction solution was extracted from the reaction slurry (reaction solution 12) and analyzed, it was confirmed that the glucose yield was 33.5 wt% and the xylose yield was 14.9 wt%, producing a total of 48.3 wt% monosaccharides. (Raw raw material EFB standard).
（３）酵素回収工程（工程（Ａ）と工程（Ｂ）の組み合わせ）
反応スラリーに、１％ＮａＯＨ水溶液を０．４５ｍｌ加えてアルカリ側にｐＨをシフトさせ、３０℃で３０分間振とうし、一部の吸着酵素を遊離させる操作を行った。振とう後の糖化反応スラリーのｐＨは６．９であった。続いて、実施例７と同様に遠心ろ過を行い、酵素回収液１２−１（ｐＨ６．９）約１０ｇと反応残渣１２約１ｇを得た。さらに、連続的酵素回収を模擬した方法で酵素回収を行った。すなわち遠心カラム中で、反応残渣１２に５０ｐｐｍのＮａＯＨ水溶液（ｐＨ９．１）を１．２ｍｌ加え、混合して３０℃で２０分間振とうした。続いて遠心ろ過を行い、酵素回収液と残渣を得た。この酵素回収操作（１．２ｍｌの５０ｐｐｍＮａＯＨ水溶液での脱着および遠心ろ過）を合計５回行い、各酵素回収液を混合して、酵素回収液１２−２を得た（合計約６ｇ）。さらに２回、酵素回収操作を繰り返し、酵素回収液１２―３を得た（合計約２ｇ）。酵素回収液のｐＨは、酵素回収操作１回目でｐＨ７．２、３回目でｐＨ７．７、５回目でｐＨ８．４、最終７回目でｐＨ８．６と漸増していた。なお得られた酵素回収液１２−１〜３には、速やかに極微量の硫酸を加え、ｐＨを５．０に調製し、保管した。
酵素回収液１２−１〜３に含まれる５種の酵素活性を測定した。回収酵素活性を表８に示す。少量のアルカリ液（１．２ｍｌ×７＝８．４ｍｌ）でも高い酵素回収率を得ることができた。
(3) Enzyme recovery step (combination of step (A) and step (B))
0.45 ml of 1% NaOH aqueous solution was added to the reaction slurry to shift the pH to the alkali side, and the mixture was shaken at 30 ° C. for 30 minutes to release a part of the adsorbing enzyme. The pH of the saccharification reaction slurry after shaking was 6.9. Subsequently, centrifugal filtration was performed in the same manner as in Example 7 to obtain about 10 g of enzyme recovery solution 12-1 (pH 6.9) and about 1 g of reaction residue 12. Furthermore, enzyme recovery was performed by a method simulating continuous enzyme recovery. That is, 1.2 ml of 50 ppm NaOH aqueous solution (pH 9.1) was added to the reaction residue 12 in a centrifugal column, mixed and shaken at 30 ° C. for 20 minutes. Subsequently, centrifugal filtration was performed to obtain an enzyme recovery solution and a residue. This enzyme recovery operation (desorption with 1.2 ml of 50 ppm NaOH aqueous solution and centrifugal filtration) was performed 5 times in total, and each enzyme recovery solution was mixed to obtain enzyme recovery solution 12-2 (total of about 6 g). Further, the enzyme recovery operation was repeated twice to obtain an enzyme recovery solution 12-3 (total of about 2 g). The pH of the enzyme recovery solution gradually increased to pH 7.2 in the first enzyme recovery operation, pH 7.7 in the third time, pH 8.4 in the fifth time, and pH 8.6 in the final seventh time. The obtained enzyme recovery solutions 12-1 to 12-3 were rapidly added with a very small amount of sulfuric acid to adjust the pH to 5.0 and stored.
Five enzyme activities contained in the enzyme recovery solutions 12-1 to 12-3 were measured. The recovered enzyme activity is shown in Table 8. Even with a small amount of alkaline solution (1.2 ml × 7 = 8.4 ml), a high enzyme recovery rate could be obtained.
（４）酵素再利用工程
実施例１と同様に、酵素回収液１２−１の限外ろ過を行った。約１０ｇの液量を約０．５ｇまで限外ろ過で濃縮し、つづいて酵素回収液１２−２を加え、液量を約１ｇまで濃縮し、さらに酵素回収液１２−３を加え、液量を約１ｇまで濃縮して、最終的に酵素回収液１２−４を得た。続いて、上記初回の糖化工程と同様の方法で、前処理ＥＦＢ−３の糖化反応を行った。ただし、添加する酵素としては、酵素回収液１２−４に初回の２０％分のフレッシュな酵素（ＣＴｅｃ６ｍｇ、ＨＴｅｃ６ｍｇ）を補充したものを使用した。４５℃、４８時間の糖化反応で、グルコース収率は３５．５ｗｔ％、キシロース収率は１３．９ｗｔ％、合計４９．４ｗｔ％（未処理の原料ＥＦＢ基準）であり、１００％フレッシュ酵素を用いた初回の反応と同等の糖収率が得られた。すなわち、酵素使用量の８０％が削減できた。
(4) Enzyme recycling step In the same manner as in Example 1, the ultrafiltration of the enzyme recovery solution 12-1 was performed. Concentrate about 10 g of liquid volume to about 0.5 g by ultrafiltration, then add enzyme recovery liquid 12-2, concentrate the liquid volume to about 1 g, and add enzyme recovery liquid 12-3. Was concentrated to about 1 g to finally obtain an enzyme recovery solution 12-4. Subsequently, a saccharification reaction of pretreatment EFB-3 was performed in the same manner as in the first saccharification step. However, as the enzyme to be added, an enzyme recovery solution 12-4 supplemented with the first 20% fresh enzyme (CTec 6 mg, HTec 6 mg) was used. In the saccharification reaction at 45 ° C. for 48 hours, the glucose yield is 35.5 wt%, the xylose yield is 13.9 wt%, and the total is 49.4 wt% (based on the raw EFB raw material), and 100% fresh enzyme is used. A sugar yield equivalent to that of the first reaction was obtained. That is, 80% of the enzyme usage was reduced.
〔比較例１〕
実施例７と同様に、前処理、糖化、酵素回収工程を行った。ただし酵素回収工程では、処理液として非特許文献７に記載の０．５Ｍのリン酸バッファー（ｐＨ７．０）を用いて、同じ脱着操作を計２回行った。得られた反応液１１、回収酵素液１１−１、２に含まれる５種の酵素活性を測定し、回収酵素活性（初期投入量に対する％で表示）を表７に示した。表７から明らかなように、０．５Ｍリン酸バッファー（ｐＨ７．０）によるｐＨを変えない脱着方法では、残渣からの酵素回収率は極めて低かった。
[Comparative Example 1]
As in Example 7, pretreatment, saccharification, and enzyme recovery steps were performed. However, in the enzyme recovery step, the same desorption operation was performed twice in total using 0.5 M phosphate buffer (pH 7.0) described in Non-Patent Document 7 as the treatment solution. Five types of enzyme activities contained in the obtained reaction solution 11 and recovered enzyme solutions 11-1 and 11-2 were measured, and the recovered enzyme activities (expressed in% relative to the initial input amount) are shown in Table 7. As is clear from Table 7, in the desorption method without changing the pH with 0.5 M phosphate buffer (pH 7.0), the enzyme recovery rate from the residue was extremely low.
なお本発明は上述した各実施形態および実施例に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。 The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention.
Claims (7)
酵素回収工程が、以下の（Ａ）および（Ｂ）の工程のうち少なくとも１つを含むことを特徴する糖化方法。
（Ａ）糖化反応スラリーにアルカリを添加して、糖化反応残渣に吸着した酵素を脱着させた後、糖化反応スラリーの固液分離を行って酵素を含有する酵素回収液を回収する工程
（Ｂ）糖化反応スラリーの固液分離により得られた糖化反応残渣にアルカリを添加して、糖化反応残渣に吸着した酵素を脱着させて酵素を含有する酵素回収液を回収し、その際、酵素回収液のｐＨが漸増するように糖化反応残渣にアルカリを添加する工程 A method for saccharification of lignocellulosic biomass comprising a saccharification step of saccharifying lignocellulosic biomass with an enzyme, and an enzyme recovery step of recovering the enzyme after completion of the saccharification step,
The saccharification method, wherein the enzyme recovery step includes at least one of the following steps (A) and (B).
(A) Step (B) of adding an alkali to the saccharification reaction slurry to desorb the enzyme adsorbed on the saccharification reaction residue and then performing solid-liquid separation of the saccharification reaction slurry to recover an enzyme recovery solution containing the enzyme. An alkali is added to the saccharification reaction residue obtained by solid-liquid separation of the saccharification reaction slurry, and the enzyme adsorbed on the saccharification reaction residue is desorbed to recover the enzyme recovery solution containing the enzyme. Adding alkali to saccharification reaction residue so that pH gradually increases
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