JP5843264B2 - Method for saccharification of potato raw material and method for producing liquid fuel - Google Patents
Method for saccharification of potato raw material and method for producing liquid fuel Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
- C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Description
本発明は、酵素を利用したジャガイモ原料の糖化方法及びその方法に基づく液体燃料の製造方法に関する。 The present invention relates to a method for saccharifying potato raw material using an enzyme and a method for producing a liquid fuel based on the method.
バイオマスから製造されるエタノール等の液体燃料は、石油を代替し、温室効果ガスの排出量削減に寄与できるため、その生産と利用が世界的に進展している。 Since liquid fuel such as ethanol produced from biomass can replace petroleum and contribute to the reduction of greenhouse gas emissions, its production and use are progressing worldwide.
ジャガイモの塊茎は水分以外はデンプンを主成分としており、このデンプンをアミラーゼ酵素で糖化し、これを発酵させてエタノールなどの液体燃料を製造することは比較的容易である。しかしジャガイモ塊茎はデンプン以外にもセルロース、ペクチン等の成分を含有しており、これらの成分を分解するのはデンプン分解に使用されるアミラーゼだけでは困難である。特にジャガイモ塊茎からデンプンを抽出した残渣（ポテトパルプ又はジャガイモデンプン抽出残渣とも呼ばれる）では、セルロース、ペクチン等の含有割合が相対的に増加して、これらを成分とする細胞壁内に残されたデンプンの利用が困難になっており、これが上記残渣の利用を制限する要因になっている。非特許文献１にはリゾプス・オリゼから分泌された酵素がポテトパルプ中のデンプンを分解して乳酸を生成することが記載されているが、リゾプス・オリゼの細胞壁分解能は低いこと（非特許文献１の表２）、また乳酸生成により糖の収率が低下してしまうことから、リゾプス・オリゼを用いた細胞壁内部のデンプンの糖化方法には依然として改良の余地がある。また、このポテトパルプの分解には複数の酵素の使用が必要であり、理想的には３種の酵素の使用が望ましいことが報告されている（非特許文献２）。しかし複数の酵素の使用はコストアップ要因であり、よりシンプルで低コストな糖化技術が求められている。
Potato tubers are mainly composed of starch except for water, and it is relatively easy to produce a liquid fuel such as ethanol by saccharifying this starch with an amylase enzyme and fermenting the starch. However, potato tubers contain components such as cellulose and pectin in addition to starch, and it is difficult to decompose these components only with amylase used for starch degradation. In particular, in the residue obtained by extracting starch from potato tubers (also called potato pulp or potato starch extraction residue), the content ratio of cellulose, pectin, etc. is relatively increased, and the starch remaining in the cell wall containing these as components Utilization has become difficult, and this is a factor that limits the utilization of the residue.
本発明は、ジャガイモ塊茎由来原料を効率的に糖化する方法、及びジャガイモ塊茎由来原料を利用した液体燃料の製造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 efficiently saccharifying a potato tuber-derived raw material and a method for producing a liquid fuel using the potato tuber-derived raw material.
本発明者らは、上記課題を解決するため鋭意検討を重ねた結果、イモ類由来原料、とりわけポテトパルプをはじめとするジャガイモ塊茎由来原料の効率的な糖化に有効な酵素群を糸状菌が産生することを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have produced filamentous fungi that are effective in efficiently saccharifying potato-derived raw materials, particularly potato tuber-derived raw materials such as potato pulp. As a result, the present invention has been completed.
すなわち、本発明は以下を包含する。
[1] ジャガイモ塊茎由来原料を、加水分解酵素を含有する細胞壁高分解性糸状菌の分泌酵素液で処理することを特徴とする、糖化物の製造方法。
[2] ジャガイモ塊茎由来原料が、ポテトパルプである、上記[1]の方法。
[3] 細胞壁高分解性糸状菌が、アクレモニウム属糸状菌又はペニシリウム属糸状菌である、上記[1]又は[2]に記載の方法。
[4] 細胞壁高分解性糸状菌が、アクレモニウム・セルロリティカス（Acremonium cellulolyticus）である、上記[1]〜[3]のいずれかの方法。
[5] 細胞壁高分解性糸状菌が、アクレモニウム・セルロリティカスTN株（受託番号FERM BP-11452）又はペニシリウムsp. NBRC 101300株である、上記[1]〜[3]のいずれかの方法。
[6] ジャガイモ塊茎由来原料を含む培地で前記糸状菌を培養することにより前記分泌酵素液を調製し、それをジャガイモ塊茎由来原料の処理に用いることを含む、上記[1]〜[5]のいずれかの方法。
[7] 上記[1]〜[6]の方法により得られた糖化物をアルコール発酵させることを特徴とする、アルコールの製造方法。
[8] サッカロミセス属酵母を用いてアルコール発酵を行う、上記[7]の方法。
[9] サッカロミセス属酵母を用いて前記糖化物をエタノール発酵させる、エタノールの製造方法である、上記[7]又は[8]の方法。
[10] ジャガイモ塊茎由来原料の糖化物を含む培地で発酵微生物を前培養し、それを用いてアルコール発酵を行う、上記[7]〜[9]の方法。
That is, the present invention includes the following.
[1] A method for producing a saccharified product, comprising treating a potato tuber-derived material with a secretory enzyme solution of a cell wall highly degradable filamentous fungus containing a hydrolase.
[2] The method of [1] above, wherein the potato tuber-derived material is potato pulp.
[3] The method according to [1] or [2] above, wherein the cell wall highly degradable filamentous fungus is Acremonium filamentous fungus or Penicillium sp.
[4] The method according to any one of [1] to [3] above, wherein the cell wall highly degradable filamentous fungus is Acremonium cellulolyticus.
[5] The method according to any of [1] to [3] above, wherein the cell wall highly degradable filamentous fungus is Acremonium cellulolyticus TN strain (Accession No. FERM BP-11452) or Penicillium sp. NBRC 101300 strain .
[6] The method according to any one of [1] to [5] above, wherein the secretory enzyme solution is prepared by culturing the filamentous fungus in a medium containing a potato tuber-derived material, and is used for the treatment of the potato tuber-derived material. Either way.
[7] A method for producing alcohol, comprising subjecting the saccharified product obtained by the methods [1] to [6] to alcohol fermentation.
[8] The method according to [7] above, wherein alcohol fermentation is performed using Saccharomyces yeasts.
[9] The method according to [7] or [8] above, which is a method for producing ethanol, wherein the saccharified product is subjected to ethanol fermentation using Saccharomyces yeast.
[10] The method according to [7] to [9] above, wherein the fermenting microorganism is pre-cultured in a medium containing a saccharified potato tuber-derived material, and alcohol fermentation is performed using the fermented microorganism.
本発明の方法によれば、ジャガイモ塊茎由来原料を効率的に加水分解し糖化することができる。 According to the method of the present invention, a potato tuber-derived raw material can be efficiently hydrolyzed and saccharified.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明の方法では、原料としてジャガイモ塊茎由来原料を用いる。ジャガイモ塊茎由来原料とは、ジャガイモの塊茎又はその処理物であって酵素処理に適した状態に加工されているものをいう。ジャガイモ塊茎由来原料は、限定するものではないが、例えば、ジャガイモ塊茎そのままの形態に加え、スライス、乱切り、角切り、つぶしたもの、破砕物、粉砕物、又は粉砕懸濁物等の任意の形態のものであってよく、皮や芽が除去されていてもいなくてもよいが、ジャガイモ塊茎の含有成分（特に、デンプン、セルロース等）をなるべく保持した状態のものが好ましい。ジャガイモ塊茎由来原料はまた、ジャガイモ塊茎を加工した際に生じる残渣であってもよく、例えば、ポテトパルプが特に好適である。ポテトパルプとは、ジャガイモ塊茎を原料としてジャガイモデンプンの抽出を行う際にジャガイモデンプン分離後の残渣として残る産物を意味する。ポテトパルプは、ジャガイモ抽出残渣とも呼ばれる。ジャガイモ塊茎由来原料は、上記のジャガイモの塊茎若しくはその処理物であって酵素処理に適した状態に加工されているもの、又はジャガイモ塊茎を加工した際に生じる残渣を、他のバイオマス原料とともに混合したものであってもよい。ジャガイモ塊茎由来原料はまた、生の状態であってもよいし、加熱された状態であってもよい。ジャガイモ塊茎由来原料は、凍結された状態であってよく、乾燥された状態であってもよく、凍結乾燥物であってもよい。ジャガイモ塊茎由来原料は殺菌処理されていてもよい。一例として、ジャガイモ塊茎由来原料について、通常微生物培養での殺菌処理に用いられる121℃、10〜30分程度のオートクレーブ処理を行ってもよい。これにより当該原料は殺菌され、以後の糖化・発酵工程での雑菌汚染を防ぐと共に、原料の分解性を高める効果も期待できるので、これは有用な処理である。 In the method of the present invention, a potato tuber-derived material is used as a material. The potato tuber-derived raw material refers to a potato tuber or a processed product thereof that has been processed into a state suitable for enzyme treatment. The potato tuber-derived raw material is not limited, but, for example, in addition to the form of the potato tuber as it is, any form such as slice, random cut, square cut, crushed, crushed, pulverized, or pulverized suspension The skin and buds may or may not be removed, but those containing the potato tuber-containing components (particularly starch, cellulose, etc.) as much as possible are preferred. The potato tuber-derived raw material may also be a residue generated when the potato tuber is processed. For example, potato pulp is particularly suitable. Potato pulp means a product remaining as a residue after separation of potato starch when potato starch is extracted from potato tubers. Potato pulp is also called potato extraction residue. The potato tuber-derived raw material is the above-mentioned potato tuber or processed product thereof, which has been processed into a state suitable for enzyme treatment, or the residue produced when processing the potato tuber is mixed with other biomass raw materials It may be a thing. The raw material from the potato tuber may also be in a raw state or in a heated state. The potato tuber-derived material may be in a frozen state, in a dried state, or in a lyophilized product. The potato tuber-derived material may be sterilized. As an example, the potato tuber-derived material may be autoclaved at 121 ° C. for about 10 to 30 minutes, which is usually used for sterilization treatment in microbial culture. This is a useful treatment because the raw material is sterilized, and it can be expected to prevent the contamination of germs in the subsequent saccharification / fermentation process and increase the degradability of the raw material.
本発明においてジャガイモ塊茎とは、一般的に食用に供するジャガイモ地下茎部分を指す。本発明で用いるジャガイモは、任意の品種のもの又はその変異種であってよいし、野生種であってもよい。 In the present invention, the potato tuber generally refers to a potato underground stem portion used for food. The potato used in the present invention may be of any varieties or variants thereof, or may be a wild species.
本発明で用いる糸状菌は、複数のバイオマス分解酵素を生産・分泌する能力を持つ真菌であって、その分泌酵素がジャガイモ塊茎の主要構成成分であるデンプン、セルロース、ペクチン等を構成糖に加水分解する能力を備えていることが好ましい。本発明で用いる糸状菌は、細胞壁高分解性糸状菌であることが好ましい。本発明において細胞壁高分解性糸状菌とは、セルロース、ペクチン等の細胞壁を構成する成分を効率良く分解することができ、それら細胞壁成分を含む例えばジャガイモ塊茎由来原料（ポテトパルプなど）を液状化するまで分解できる糸状菌をいう。例えばアナモルフであるアクレモニウム属又はペニシリウム属に属する糸状菌が、本発明で用いる好適な糸状菌として挙げられる。本発明で用いる糸状菌として好ましい例としては、アクレモニウム・セルロリティカス（Acremonium cellulolyticus）が挙げられる。本発明で用いる糸状菌の特に好ましい例は、アクレモニウム・セルロリティカス（Acremonium cellulolyticus）TN株、及びペニシリウム（Penicillium）sp. NBRC 101300株である。 The filamentous fungus used in the present invention is a fungus having the ability to produce and secrete a plurality of biomass degrading enzymes, and the secreted enzyme hydrolyzes starch, cellulose, pectin, etc., which are the main components of potato tubers, into constituent sugars. It is preferable to have the ability to do this. The filamentous fungus used in the present invention is preferably a cell wall highly degradable filamentous fungus. In the present invention, the cell wall highly degradable filamentous fungus can efficiently decompose components constituting the cell wall such as cellulose and pectin, and liquefy potato tuber-derived raw materials (potato pulp, etc.) containing these cell wall components. A filamentous fungus that can be degraded to For example, filamentous fungi belonging to the genus Acremonium or Penicillium, which are anamorphs, can be mentioned as suitable filamentous fungi used in the present invention. A preferable example of the filamentous fungus used in the present invention is Acremonium cellulolyticus. Particularly preferred examples of the filamentous fungus used in the present invention are Acremonium cellulolyticus TN strain and Penicillium sp. NBRC 101300 strain.
各種糸状菌は、市販品、又はカルチャーコレクション若しくはブダペスト条約に基づく寄託機関から入手することができる。 Various filamentous fungi can be obtained from commercial products or from depository institutions based on the Culture Collection or the Budapest Treaty.
Acremonium cellulolyticus TN株は、２０１２年１月１２日付けで独立行政法人産業技術総合研究所特許生物寄託センター（日本国茨城県つくば市東１丁目１番１ 中央第６）に、受託番号FERM BP-11452の下でブタペスト条約に基づき国際寄託されている。 Acremonium cellulolyticus TN strain was transferred to the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (January 1-1 1-1 Tsukuba, Ibaraki, Japan, Central 6th) on January 12, 2012, with the accession number FERM BP-11452 Has been deposited internationally under the Budapest Treaty.
Penicillium sp. NBRC 101300株は、独立行政法人製品評価技術基盤機構（NITE）のNBRC（NITE Biological Resource Center)（日本）のカタログ［NBRC Catalogue of Biological Resources, Microorganisms, Microorganism-Related DNA Resources, Human-Related DNA Resources, Second Edition (2010)］に収載されており、番号101300の下でNBRCから入手することができる。 Penicillium sp. NBRC 101300 is a catalog of NBRC (NITE Biological Resource Center) of Japan (NITE) [NBRC Catalog of Biological Resources, Microorganisms, Microorganism-Related DNA Resources, Human-Related DNA Resources, Second Edition (2010)] and is available from NBRC under the number 101300.
本発明の方法では、ジャガイモ塊茎由来原料の処理に、前記糸状菌が産生する加水分解酵素を含有する分泌酵素液を用いる。本発明において糸状菌の分泌酵素液とは、糸状菌を培養することにより生産され培地中に分泌される分泌酵素群を含む液（酵素液）をいう。糸状菌のこの分泌酵素液は、デンプン、セルロース、ペクチン等のジャガイモが含有する多糖類を分解する能力を有する酵素（加水分解酵素）を一種又は複数種、好ましくは複数種含み、少なくともセルラーゼを含む。本発明に係る糸状菌の分泌酵素液は、典型的には、セルラーゼ（セルロース加水分解酵素）、ペクチナーゼ（ペクチン加水分解酵素）、及びアミラーゼ（デンプン加水分解酵素）を含む。本発明に係る糸状菌の分泌酵素液は、ガラクタン分解酵素をさらに含むことも好ましい。なお本明細書では、複数種の酵素を含む分泌酵素液を、酵素系と表現することがある。これらの酵素は糸状菌が細胞内で生産し、生産後細胞外に分泌される。この分泌酵素液は、糸状菌培養物又は培養上清でありうる。前記糸状菌の分泌酵素液は、一種以上（好ましくは１種）の糸状菌を炭素源の存在下で培養して加水分解酵素の生産を誘導し、その培養液中に当該酵素を分泌させることにより調製することができる。糸状菌を培養する培地は、糸状菌の培養に適した任意の培養培地に炭素源等を加えて調製することができる。糸状菌を培養するために必要な培地中の炭素源としては任意の有機物を使用することができるが、ジャガイモ塊茎由来原料（例えばポテトパルプ）又はその酵素分解物（例えば糖化物）を炭素源として用いることがセルラーゼ、ペクチナーゼ及びアミラーゼ等の加水分解酵素の生産を誘導する上でより好適である。後者の場合、他の炭素源を用いた場合と比べ、ジャガイモ塊茎に特に多く存在する成分（デンプン、セルロース、ペクチン等）に対する分解性が優れた酵素を生産できる。このため、糸状菌を培養する培地は、炭素源としてデンプン、セルロース及びペクチンのうち少なくとも１つ（好ましくは全部）を含むことが好ましい。糸状菌を培養する培地は、ジャガイモ塊茎由来原料（例えばポテトパルプ）に加えて、その酵素分解物（例えば糖化物）やジャガイモ塊茎由来原料以外の炭素源（例えば、粉末セルロース等の精製セルロースや精製デンプンなど）を含むことも好ましい。ジャガイモ塊茎由来原料以外の炭素源は、例えば0.5％以上、好ましくは1.5％以上、さらに好ましくは5％以上、例えば0.5〜30％の濃度でジャガイモ塊茎由来原料に添加することができる。ジャガイモ塊茎由来原料に加えて他の炭素源を添加することにより、セルラーゼ、ペクチナーゼ及びアミラーゼ等の加水分解酵素の生産をさらに効果的に誘導できる。 In the method of the present invention, a secretory enzyme solution containing a hydrolase produced by the filamentous fungus is used for processing the potato tuber-derived material. In the present invention, the secretory enzyme solution of filamentous fungi refers to a solution (enzyme solution) containing a secretory enzyme group produced by culturing filamentous fungi and secreted into the medium. This secretory enzyme solution of filamentous fungi contains one or more, preferably several, enzymes having the ability to degrade polysaccharides contained in potato such as starch, cellulose, pectin, etc., and preferably contains at least cellulase. . The secretory enzyme solution of the filamentous fungus according to the present invention typically contains cellulase (cellulose hydrolase), pectinase (pectin hydrolase), and amylase (starch hydrolase). The secretory enzyme solution of the filamentous fungus according to the present invention preferably further contains a galactan degrading enzyme. In the present specification, a secretory enzyme solution containing a plurality of types of enzymes may be expressed as an enzyme system. These enzymes are produced intracellularly by filamentous fungi and secreted extracellularly after production. This secreted enzyme solution may be a filamentous fungus culture or a culture supernatant. The filamentous fungus secretory enzyme solution is obtained by culturing one or more (preferably one) filamentous fungi in the presence of a carbon source to induce hydrolase production, and secreting the enzyme into the culture solution. Can be prepared. A medium for culturing filamentous fungi can be prepared by adding a carbon source or the like to any culture medium suitable for culturing filamentous fungi. Arbitrary organic substances can be used as a carbon source in the medium necessary for culturing filamentous fungi, but potato tuber-derived raw materials (for example, potato pulp) or enzyme degradation products (for example, saccharified products) are used as the carbon source. It is more preferable to use it for inducing the production of hydrolases such as cellulase, pectinase and amylase. In the latter case, it is possible to produce an enzyme having excellent degradability with respect to components (starch, cellulose, pectin, etc.) that are particularly abundant in potato tubers as compared with the case of using other carbon sources. For this reason, it is preferable that the culture medium which culture | cultivates a filamentous fungus contains at least 1 (preferably all) among starch, a cellulose, and pectin as a carbon source. The medium for culturing the filamentous fungus is not only potato tuber-derived raw material (for example, potato pulp), but also its enzymatic degradation product (for example, saccharified product) or a carbon source other than the potato tuber-derived raw material (for example, purified cellulose such as powdered cellulose or purified It is also preferable to include starch. The carbon source other than the potato tuber-derived raw material can be added to the potato tuber-derived raw material at a concentration of 0.5% or more, preferably 1.5% or more, more preferably 5% or more, for example 0.5 to 30%. By adding other carbon sources in addition to the potato tuber-derived raw material, the production of hydrolases such as cellulase, pectinase and amylase can be more effectively induced.
糸状菌の分泌酵素液は、そのような糸状菌の培養液から（例えば培養上清の形態で）採取、分離、又は精製して使用してもよいし、採取、分離、又は精製することなく培養液をそのまま、ジャガイモ塊茎由来原料の処理に使用することもできる。糸状菌の分泌酵素液として、工業的に生産され市販されている製品（例えば、アクレモニウムセルラーゼ（Meiji Seika ファルマ株式会社製））を使用してもよい。 The secretory enzyme solution of the filamentous fungus may be used after being collected, separated or purified from the culture solution of such a filamentous fungus (for example, in the form of a culture supernatant) or without being collected, separated or purified. The culture solution can be used as it is for the processing of the potato tuber-derived material. An industrially produced and marketed product (for example, Acremonium cellulase (manufactured by Meiji Seika Pharma Co., Ltd.)) may be used as a secretory enzyme solution for filamentous fungi.
本発明の方法では、ジャガイモ塊茎由来原料、又はジャガイモ塊茎由来原料を含む溶液又は液体培地に、糸状菌の分泌酵素液を添加して反応させることにより、酵素処理を行うことができる。 In the method of the present invention, the enzyme treatment can be performed by adding and reacting a secretory enzyme solution of a filamentous fungus to a potato tuber-derived material or a solution or liquid medium containing a potato tuber-derived material.
あるいは本発明の方法では、ジャガイモ塊茎由来原料、又はジャガイモ塊茎由来原料を含む液体培地中で、本発明の糸状菌を培養してセルラーゼ等の加水分解酵素の生産を誘導し、培地中に分泌させて分泌酵素液とし、そこにジャガイモ塊茎由来原料をさらに加えて反応させることにより、酵素処理を行ってもよい。 Alternatively, in the method of the present invention, the filamentous fungus of the present invention is cultured in a liquid medium containing a potato tuber-derived raw material or a potato tuber-derived raw material to induce the production of a hydrolase such as cellulase and secreted into the medium. Then, the enzyme treatment may be performed by preparing a secreted enzyme solution and further adding a potato tuber-derived raw material to the reaction.
酵素処理は各酵素の至適温度、pHで行われることが好ましいが、例えばアクレモニウム・セルロリティカスが生産する酵素系の場合、40〜60℃、pH4〜6程度、好ましくは45〜55℃、pH4.5〜5.5で行われるのが適当である。
The enzyme treatment is preferably performed at the optimum temperature and pH of each enzyme. For example, in the case of an enzyme system produced by Acremonium cellulolyticus, 40 to 60 ° C., about
酵素処理は、ジャガイモ塊茎由来原料（好適には、ポテトパルプ）を5％以上、好ましくは7％以上、より好ましくは10％以上、さらに好ましくは20％以上、なお好ましくは30％以上、例えば50％以上100％以下の濃度、例えば5〜50％の濃度で含有する溶液又は培地を用いて行うことが好ましい。 In the enzyme treatment, the potato tuber-derived material (preferably potato pulp) is 5% or more, preferably 7% or more, more preferably 10% or more, still more preferably 20% or more, still more preferably 30% or more, for example 50 It is preferable to use a solution or a medium containing a concentration of not less than 100% and not more than 100%, such as a concentration of 5 to 50%.
以上のような酵素反応により、ジャガイモ塊茎を構成する多糖類（セルロース、デンプン、ペクチン等）が加水分解されて単糖類又は少糖類（例えば、グルコース、ガラクトース、マルトース、セロビオース等）が生成される（糖化反応）。本発明は、このような、ジャガイモ塊茎由来原料からの糖化物の製造方法も提供する。本発明はまた、このような糖化反応による、ジャガイモ塊茎由来原料の糖化方法も提供する。 By the enzyme reaction as described above, polysaccharides (cellulose, starch, pectin, etc.) constituting potato tubers are hydrolyzed to produce monosaccharides or oligosaccharides (eg, glucose, galactose, maltose, cellobiose, etc.) ( Saccharification reaction). The present invention also provides a method for producing a saccharified product from such potato tuber-derived materials. The present invention also provides a method for saccharification of a potato tuber-derived raw material by such a saccharification reaction.
このようにしてジャガイモ塊茎由来原料を糖化して得られる糖化物（典型的には、糖化液）は、発酵させることにより、様々な有用物質に変換することができる。特に、アルコール発酵により、糖化物中の単糖類又は少糖類からアルコールを生産することができる。得られるアルコールは例えば液体燃料や工業材料等として使用することができる。 Thus, the saccharified product (typically, saccharified solution) obtained by saccharifying the potato tuber-derived raw material can be converted into various useful substances by fermentation. In particular, alcohol can be produced from monosaccharides or oligosaccharides in a saccharified product by alcohol fermentation. The alcohol obtained can be used, for example, as a liquid fuel or industrial material.
アルコール発酵は、アルコール発酵を行う発酵微生物、例えば酵母を用いて行うことができる。酵母としては、限定するものではないが、例えば、サッカロミセス・セルビシエ（Saccharomyces cerevisiae）、サッカロミセス・パストリアヌス（Saccharomyces pastorianus）等のサッカロミセス属（Saccharomyces）菌、シゾサッカロミセス・ポンベ（Saccharomyces pombe)）等のシゾサッカロミセス属（Schizosaccharomyces）菌、クリュイベロミセス・マルシアヌス（Kluyveromyces marxianus）等のクリュイベロミセス属（Kluyveromyces）菌、イサトケンキア・オリエンタリス（Issatchenkia orientalis）等のイサトケンキア属（Issatchenkia）菌、等が挙げられる。 Alcohol fermentation can be performed using a fermentation microorganism that performs alcohol fermentation, such as yeast. Examples of yeast include, but are not limited to, for example, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces pastorianus (Saccharomyces), Saccharomyces pombe (Saccharomyces pombe, etc.) Schizosaccharomyces bacteria, Kluyveromyces marxianus and other Kluyveromyces bacteria, Isatokenchia orientalis, etc.
例えば酵母としてエタノール発酵能を有するサッカロミセス属酵母を用いることにより、上記糖化物をエタノール発酵させることができる。とりわけサッカロミセス・セルビシエが好ましく、これを糖化物に加えて培養することにより、グルコース等の単糖類からエタノールを生産（製造）することができる。このエタノールはガソリンに代替できる液体燃料として使用できる。サッカロミセス・セルビシエによる発酵実験により、ジャガイモ塊茎由来原料の糖化物は、酵母への阻害効果が見られず、良好な発酵原料として利用できることが示されている。本発明において「発酵微生物」とは発酵を行う能力を有する微生物をいう。 For example, the saccharified product can be ethanol-fermented by using yeast of the genus Saccharomyces having ethanol fermentation ability as yeast. In particular, Saccharomyces cerevisiae is preferable, and ethanol can be produced (manufactured) from monosaccharides such as glucose by adding it to a saccharified product and culturing. This ethanol can be used as a liquid fuel that can replace gasoline. Fermentation experiments with Saccharomyces cerevisiae show that the saccharified potato tuber-derived raw material does not show an inhibitory effect on yeast and can be used as a good fermentation raw material. In the present invention, “fermenting microorganism” refers to a microorganism having the ability to perform fermentation.
上記の発酵はサッカロミセス・セルビシエを用いてエタノールを生成するアルコール発酵に限定されるわけではなく、用いる発酵微生物に応じて、ブタノール等の他のアルコールを製造することもできる。そのような各種アルコールも液体燃料等に利用することができる。 The above fermentation is not limited to alcohol fermentation using Saccharomyces cerevisiae to produce ethanol, and other alcohols such as butanol can be produced depending on the fermentation microorganism used. Such various alcohols can also be used for liquid fuel and the like.
ジャガイモ塊茎由来原料を糖化して得られる糖化物を発酵させることにより、アルコール以外の物質、例えば脂肪酸等の液体燃料成分となりうる有用物質も生産することができる。この発酵は、対応する発酵微生物を用いることにより達成される。 By fermenting a saccharified product obtained by saccharifying a potato tuber-derived raw material, a useful substance that can be a liquid fuel component such as a substance other than alcohol, such as a fatty acid, can also be produced. This fermentation is achieved by using the corresponding fermenting microorganism.
発酵微生物は、糖化物の発酵に用いる前に、一定の量まで増殖させて準備する工程（一般に前培養と呼ばれる）を行うことが好ましい。本発明の方法では、ジャガイモ塊茎由来原料の糖化物を含む培地で発酵微生物を前培養し、それを用いて糖化物の発酵（好ましくはアルコール発酵）を行うことが好ましい。発酵は当該発酵微生物の通常の発酵条件下で行えばよく、例えば嫌気性条件下で行っても好気性条件下で行ってもよい。サッカロミセス・セルビシエの場合には、嫌気性の発酵条件下で行うことが好ましい。 It is preferable that the fermenting microorganism is subjected to a step (generally referred to as preculture) in which it is grown and prepared to a certain amount before being used for fermentation of a saccharified product. In the method of the present invention, it is preferable to pre-culture fermented microorganisms in a medium containing a potato tuber-derived raw material saccharified product, and to perform saccharified product fermentation (preferably alcoholic fermentation) using it. Fermentation may be performed under normal fermentation conditions of the fermenting microorganism. For example, the fermentation may be performed under anaerobic conditions or aerobic conditions. In the case of Saccharomyces cerevisiae, it is preferably carried out under anaerobic fermentation conditions.
本発明における前培養では、ジャガイモ塊茎由来原料の糖化物を含む培地、例えば、ジャガイモ塊茎由来原料の糖化液それ自体であってもよいし、ジャガイモ塊茎由来原料の糖化物を培養培地等の他の培地に加えて調製してもよい。ジャガイモ塊茎由来原料の糖化物を用いることにより、一般的に用いられる栄養源である廃糖蜜を使用した場合と比べて微生物のより良好な増殖を得ることができる。このようにしてジャガイモ塊茎由来原料の糖化物で前培養した微生物を、本培養としての糖化物の発酵に用いることにより、アルコール発酵を非常に効率良く行うことができる。なおジャガイモ塊茎由来原料の糖化物で前培養した微生物は、ジャガイモ塊茎由来原料の糖化物の発酵だけでなく、他の各種原料を用いた発酵においても広く使用可能である。 In the pre-culture in the present invention, a medium containing a saccharified product of potato tuber-derived raw material, for example, a saccharified solution of a potato tuber-derived raw material itself may be used, or a saccharified product of a potato tuber-derived raw material may be used as a culture medium or the like. It may be prepared in addition to the medium. By using the saccharified material derived from the potato tuber, better growth of the microorganisms can be obtained as compared with the case where waste molasses, which is a commonly used nutrient source, is used. Thus, alcohol fermentation can be performed very efficiently by using the microorganisms pre-cultured with the saccharified material derived from the potato tuber for the fermentation of the saccharified product as the main culture. In addition, the microorganisms pre-cultured with the saccharified material derived from the potato tuber can be widely used not only in fermentation of the saccharified material derived from the potato tuber but also in fermentation using other various materials.
従来、ジャガイモ塊茎由来原料、特にポテトパルプ等については、その成分を総合的に効率良く分解・糖化できる技術が存在せず、その利用に対する障害になっていた。本発明の方法では、糸状菌由来の酵素系を用いることにより、ジャガイモ塊茎由来原料に含まれるセルロース、デンプン、ペクチンの主要成分を効率良く分解でき、その結果、発酵によって液体燃料に変換可能である単糖類を、効率良く得ることが可能である。本発明の方法によって、従来困難であったジャガイモ塊茎に由来する原料の総合的な分解が容易にできるようになり、これを発酵させることでアルコールを始めとする液体燃料の製造が可能となることで、従来利用度の低かったバイオマス原料の有効活用法を提供することができる。 Conventionally, for potato tuber-derived raw materials, particularly potato pulp and the like, there has been no technology capable of comprehensively and efficiently decomposing and saccharifying the components, which has been an obstacle to their use. In the method of the present invention, by using an enzyme system derived from a filamentous fungus, the main components of cellulose, starch and pectin contained in the raw material derived from potato tubers can be efficiently decomposed, and as a result, can be converted into liquid fuel by fermentation. Monosaccharides can be obtained efficiently. The method of the present invention makes it possible to easily decompose the raw materials derived from potato tubers, which has been difficult in the past, and to produce liquid fuels including alcohol by fermenting them. Thus, it is possible to provide an effective utilization method of biomass raw materials that have been low in utilization.
次に実施例を挙げ、本発明を更に詳しく説明するが、本発明はこれらに限定されるものではない。 EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not limited to these.
［実施例１］
（１）ジャガイモデンプン搾り粕基本成分の分析
中国黒龍江省北大荒馬鈴薯集団有限公司のデンプン工場で採取されたポテトパルプ（ジャガイモデンプン搾り粕）を凍結乾燥したものを入手して後述の糖化試験に使用した。
[Example 1]
(1) Analysis of the basic components of potato starch squeezed potato freeze-dried potato pulp (potato starch squeezed potato) collected at the starch factory of Kita Ara potato Group Co., Ltd., Heilongjiang Province, China for saccharification tests described below used.
そこでまず、使用したポテトパルプの成分組成を常法により分析した。その結果を表１に示す。 Therefore, first, the component composition of the potato pulp used was analyzed by a conventional method. The results are shown in Table 1.
なお、表１の下部には上記ポテトパルプ中の多糖を完全分解した後の単糖組成を示した。この単糖組成は、ポテトパルプを硫酸で完全分解して得られた実測値である。グルコースはほとんどがデンプンとセルロースの分解から生成するため、デンプンとセルロースの量の合計が単糖組成中のグルコール含量とほぼ一致する。 The lower part of Table 1 shows the monosaccharide composition after complete degradation of the polysaccharide in the potato pulp. This monosaccharide composition is an actual measurement value obtained by completely decomposing potato pulp with sulfuric acid. Since glucose is mostly produced from the degradation of starch and cellulose, the total amount of starch and cellulose is approximately consistent with the glucose content in the monosaccharide composition.
（２）酵素を用いた糖化
上記（１）の凍結乾燥ポテトパルプ（試料）に水を加え、濃度を10％、20％、30％（各w/v）とした。これに、市販セルラーゼ製剤アクレモニウムセルラーゼ（Meiji Seika ファルマ株式会社製；Acremonium cellulolyticusの生産するセルラーゼ）を10 Filter Paper Unit（FPU；濾紙分解活性）/g基質の割合で加えて、0.05Mのクエン酸バッファー中で、50℃にて24時間又は48時間反応を行い、生成したグルコース、ガラクトースの量を測定した。グルコース及びガラクトースの測定は、アミネックスHPX-87H(BioRad 社)カラムを装着した日本分光社製高速液体クロマトグラフィーシステム（検出器：示差屈折計2031Plus）を用いて、それぞれの標品を基準にして定量した。その結果を図１に示す。図１に示されるようにグルコース及びガラクトースの生成量は用いた試料の濃度に依存して増加した。酵素処理により当初ゲル状であったポテトパルプは分解して液状化し、グルコース、ガラクトースの単糖が生成された。
一方、同じく市販セルラーゼ製剤であるAccellerase 1000（Genencore社製；Trichoderma reesei由来）を用いて同様に（１）のポテトパルプを酵素処理する実験を行ったところ、10％濃度でも残渣が液状化せず、分解がみられなかった。
(2) Saccharification using enzyme Water was added to the freeze-dried potato pulp (sample) of (1) above to adjust the concentration to 10%, 20%, and 30% (each w / v). To this was added a commercially available cellulase preparation Acremonium cellulase (manufactured by Meiji Seika Pharma Co., Ltd .; cellulase produced by Acremonium cellulolyticus) at a ratio of 10 Filter Paper Unit (FPU; filter paper degradation activity) / g substrate, and 0.05M citric acid The reaction was performed in a buffer at 50 ° C. for 24 hours or 48 hours, and the amounts of produced glucose and galactose were measured. Glucose and galactose are measured using a high-performance liquid chromatography system (detector: differential refractometer 2031Plus) manufactured by JASCO Corporation equipped with an Aminex HPX-87H (BioRad) column. did. The result is shown in FIG. As shown in FIG. 1, the production amounts of glucose and galactose increased depending on the concentration of the sample used. The potato pulp that was initially gelled by enzymatic treatment was decomposed and liquefied, producing glucose and galactose monosaccharides.
On the other hand, when Accelerase 1000 (Genencore; Trichoderma reesei origin) was similarly used for the enzyme treatment of the potato pulp of (1), the residue was not liquefied even at 10% concentration. No decomposition was observed.
この結果から、イモ類、特にジャガイモ由来原料の糖化にアクレモニウム属菌の生産するセルラーゼは非常に適していることが示された。 From this result, it was shown that cellulase produced by Acremonium spp. Is very suitable for saccharification of potatoes, particularly potato-derived raw materials.
［実施例２］
（１）ポテトパルプを用いた酵素生産
セルラーゼ生産菌Acremonium cellulolyticus TN株（受託番号：FERM BP-11452）をポテトパルプを炭素源として培養して、酵素を生産させた。また対照として、通常セルラーゼ生産の炭素源として用いられる粉末セルロースを、ポテトパルプの代わりに炭素源として用いた培養も行った。
[Example 2]
(1) Enzyme production using potato pulp Cellulase-producing bacteria Acremonium cellulolyticus TN strain (accession number: FERM BP-11452) was cultured using potato pulp as a carbon source to produce an enzyme. As a control, culturing was also performed using powdered cellulose, which is usually used as a carbon source for cellulase production, as a carbon source instead of potato pulp.
培養に用いた培地の組成は以下のとおり：24 g/L KH2PO4、1 g/L Tween 80、5 g/L (NH4)2SO4、1.2 g/L MgSO4・7H2O、0.01 g/L ZnSO4・7H2O、0.01 g/L MnSO4・6H2O、0.01 g/L CuSO47H2O、4 g/L 尿素、4.7 g/L 酒石酸カリウム一水和物、及び炭素源として粉末セルロース（Solka Floc（登録商標）；対照）又は凍結乾燥ポテトパルプ（5％、7％、10％）。
The composition of the medium used for the culture is as follows: 24 g / L KH 2 PO 4 , 1 g /
培養は、反応温度30℃、撹拌速度200rpmで行った。前培養を粉末セルロースを炭素源として行い、前培養開始から72時間後、前培養液を5％になるように培地に添加して、本培養を行った。 The culture was performed at a reaction temperature of 30 ° C. and a stirring speed of 200 rpm. Pre-culture was performed using powdered cellulose as a carbon source, and after 72 hours from the start of pre-culture, the pre-culture was added to the medium so as to be 5%, and main culture was performed.
本培養の培養開始から5日後、7日後、14日後の培地を採取し、培地中に分泌された酵素セルラーゼの活性を測定した。セルラーゼ活性の測定は、ろ紙（Whatman No.1）を基質に、50 mM クエン酸緩衝液（pH 5.0）中で50℃で60分間反応させ、生成した還元糖の量をジニトロサリチル酸法により比色して定量して行い、活性をFPUで表した（図２）。 After 5 days, 7 days, and 14 days from the start of the main culture, the medium was collected, and the activity of the enzyme cellulase secreted into the medium was measured. Cellulase activity was measured using a filter paper (Whatman No. 1) as a substrate, reacted in 50 mM citrate buffer (pH 5.0) at 50 ° C. for 60 minutes, and the amount of produced reducing sugar was calorimetrically determined by the dinitrosalicylic acid method. The activity was expressed in FPU (FIG. 2).
図２に示すとおり、粉末セルロースを炭素源にした場合より活性の濃度は低いものの、ポテトパルプを炭素源とした培養によりセルラーゼ活性を持つ酵素を生産可能であった。 As shown in FIG. 2, an enzyme having cellulase activity could be produced by culturing with potato pulp as a carbon source, although the concentration of activity was lower than when powdered cellulose was used as the carbon source.
さらに、凍結乾燥ポテトパルプ（5％）に0.5％、1.5％、又は5％粉末セルロースを添加したものを炭素源として、上記と同様にAcremonium cellulolyticus （A. cellulolyticus）TN株の培養を行い、酵素を生産させた。培養後の培地について上記と同様にしてセルラーゼ活性（FPU活性）を測定したところ、ポテトパルプへのセルロース添加量に応じたFPU活性の向上が認められた（図３）。 Furthermore, using the addition of 0.5%, 1.5%, or 5% powdered cellulose to freeze-dried potato pulp (5%), the Acremonium cellulolyticus (A. cellulolyticus) TN strain is cultured in the same manner as described above, and the enzyme Was produced. When the cellulase activity (FPU activity) was measured in the same manner as described above for the cultured medium, an improvement in FPU activity according to the amount of cellulose added to the potato pulp was observed (FIG. 3).
（２）ポテトパルプを炭素源として生産した酵素によるポテトパルプの糖化
本実施例の上記（１）においてポテトパルプを炭素源としてA. cellulolyticus TN株を培養して得られた培養物の培養上清を酵素液として採取し、これを限外濾過膜付チューブ（VIVASPIN 6、Sartorius社）を用いて限外濾過及び濃縮した。これを各5 FPU/gの割合で、30％（w/v）ポテトパルプに添加し、実施例１−（２）と同様にして糖化を24時間又は48時間にわたり行った。
(2) Saccharification of potato pulp by enzyme produced using potato pulp as carbon source Culture supernatant of culture obtained by culturing A. cellulolyticus TN strain using potato pulp as carbon source in (1) of this example Was collected as an enzyme solution, and this was ultrafiltered and concentrated using a tube with an ultrafiltration membrane (
同様に、本実施例の上記（１）において粉末セルロースを炭素源としてA. cellulolyticus TN株を培養して得られた培養物の培養上清を酵素液として採取し、上記と同様の反応条件で糖化試験を行った。また対照として、実施例１で使用した市販セルラーゼ製剤アクレモニウムセルラーゼを用いた糖化試験も同様の反応条件で実施した。 Similarly, the culture supernatant of the culture obtained by culturing A. cellulolyticus TN strain using powdered cellulose as a carbon source in the above (1) of this example was collected as an enzyme solution, and the reaction conditions were the same as above. A saccharification test was performed. As a control, a saccharification test using the commercially available cellulase preparation Acremonium cellulase used in Example 1 was also carried out under the same reaction conditions.
次いで、得られた糖化液についてグルコース及びガラクトースの量を実施例１と同様にしてそれぞれ測定した。 Next, the amounts of glucose and galactose were measured in the same manner as in Example 1 for the obtained saccharified solution.
以上の結果を図４に示す。図４に示すとおり、ポテトパルプを炭素源としてA. cellulolyticus TN株により生産した酵素液を用いて、ポテトパルプからグルコース及びガラクトースを生産することができた。また粉末セルロースを炭素源として生産した酵素液又は市販セルラーゼ製剤アクレモニウムセルラーゼを用いた場合にもポテトパルプからグルコース及びガラクトースを生産することができた。特にポテトパルプを炭素源として生産した酵素液を用いた場合、粉末セルロースを炭素源として生産した酵素液又は市販セルラーゼ製剤アクレモニウムセルラーゼを用いた場合を上回る糖収量を得ることができた。ガラクトースについては、粉末セルロースを炭素源として生産した酵素液に比べてポテトパルプを炭素源として得た酵素液で顕著に高い収量が得られた。このことから、ポテトパルプを炭素源とした場合、ガラクタン分解酵素の生産が誘導され、得られた分泌酵素液中にガラクタン分解酵素が含まれていたと考えられる。 The above results are shown in FIG. As shown in FIG. 4, glucose and galactose could be produced from potato pulp using an enzyme solution produced by A. cellulolyticus TN strain using potato pulp as a carbon source. Glucose and galactose could also be produced from potato pulp when an enzyme solution produced using powdered cellulose as a carbon source or a commercially available cellulase preparation Acremonium cellulase was used. In particular, when an enzyme solution produced using potato pulp as a carbon source was used, a sugar yield higher than that obtained using an enzyme solution produced using powdered cellulose as a carbon source or the commercially available cellulase preparation Acremonium cellulase could be obtained. For galactose, a significantly higher yield was obtained with the enzyme solution obtained using potato pulp as the carbon source than the enzyme solution produced using powdered cellulose as the carbon source. From this, when potato pulp is used as a carbon source, it is considered that the production of galactan-degrading enzyme was induced and the resulting secretory enzyme solution contained galactan-degrading enzyme.
［実施例３］
A. cellulolyticus TN株の代わりに、糸状菌Penicillium sp. NBRC 101300株（独立行政法人製品評価技術基盤機構（NITE）のNBRCのカタログ［NBRC Catalogue of Biological Resources, Microorganisms, Microorganism-Related DNA Resources, Human-Related DNA Resources, Second Edition (2010)］に収載されており、NBRCから入手した）、Trichoderma reesei RUT C-30株（the American Type Culture Collection （ATCC）no. 56765））を、実施例２と同様の条件で、ポテトパルプを炭素源として用いて培養し、得られた酵素液を使用してポテトパルプを糖化させた。糖化液についてグルコースとガラクトースの量を測定した結果を図５に示す。
[Example 3]
Instead of the A. cellulolyticus TN strain, the filamentous fungus Penicillium sp. NBRC 101300 strain (NBRC Catalog of Biological Resources, Microorganisms, Microorganism-Related DNA Resources, Human- Related DNA Resources, Second Edition (2010)], obtained from NBRC), and Trichoderma reesei RUT C-30 strain (the American Type Culture Collection (ATCC) no. 56765)) as in Example 2. Under these conditions, potato pulp was cultured as a carbon source, and the resulting enzyme solution was used to saccharify the potato pulp. The result of having measured the quantity of glucose and galactose about a saccharified liquid is shown in FIG.
この結果、3種の糸状菌はいずれもグルコースとガラクトースを生産したことが示された。図５に示すとおり、A. cellulolyticus由来酵素液を用いた場合のグルコース量が最も高く、糖化において特に好適であることが示された。しかしながらPenicillium sp.由来の酵素液についても、A. cellulolyticus由来酵素液の約75％のグルコース量が得られており、またガラクトース量については、A. cellulolyticusよりも高い収量が得られたことから、この菌も糖化に好適であることが示された。これらの菌は、特に、ポテトパルプの糖化用の酵素生産に有用である。 As a result, it was shown that all three fungi produced glucose and galactose. As shown in FIG. 5, the amount of glucose in the case of using an A. cellulolyticus-derived enzyme solution was the highest, indicating that it is particularly suitable for saccharification. However, the enzyme solution derived from Penicillium sp. Also has a glucose amount of about 75% of the enzyme solution derived from A. cellulolyticus, and the galactose amount was higher than that of A. cellulolyticus, This bacterium was also shown to be suitable for saccharification. These fungi are particularly useful for producing enzymes for saccharification of potato pulp.
セルラーゼ生産菌として工業的に一般に使用されているT. reesei由来酵素液では、残渣が液状化して糖化が開始されるのに40時間以上を要し、その反応はA. cellulolyticus TN株及びPenicillium sp. NBRC 101300株よりも遅かった。 In the enzyme solution derived from T. reesei that is generally used industrially as a cellulase-producing bacterium, it takes 40 hours or more for the residue to liquefy and start saccharification, and the reaction takes place in A. cellulolyticus TN strain and Penicillium sp. It was slower than NBRC 101300 shares.
［実施例４］
（１）ジャガイモ糖化液による発酵微生物の前培養
糖化液の発酵に用いる微生物を一定量まで増やしておくための前培養に、栄養源としてジャガイモ糖化液を使用できるかどうかを試験した。
[Example 4]
(1) Pre-culture of fermented microorganisms with potato saccharified liquid It was tested whether potato saccharified liquid can be used as a nutrient source for pre-culture to increase the number of microorganisms used for fermentation of saccharified liquid to a certain amount.
酵母Saccharomyces cerevisiae IR-2株（独立行政法人産業技術総合研究所特許生物寄託センターに寄託。受託番号FERM BP-754）をYPD培地（1％ 酵母エキス、1％ ポリペプトン、2％ グルコース）で一晩培養し、OD600値（Optical density：吸光度による菌体細胞数を表す指標）が0.1になるように培地に添加し、30℃、攪拌速度120 rpmで培養実験を行った。 Yeast Saccharomyces cerevisiae IR-2 strain (deposited at National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center. Accession number FERM BP-754) overnight in YPD medium (1% yeast extract, 1% polypeptone, 2% glucose) The cells were cultured and added to the medium so that the OD 600 value (Optical density: an index representing the number of bacterial cells by absorbance) was 0.1, and a culture experiment was performed at 30 ° C. and a stirring speed of 120 rpm.
培地はYPD培地をベースとし、ポテトパルプを実施例１−（２）で市販酵素アクレモニウムセルラーゼで処理して得た糖化液（ジャガイモ糖化液）、及び中国で入手したサトウダイコンの廃糖蜜を使用して調製した。対照の培地としてはYPD培地を用いた。培養時間は6時間、18時間、又は24時間とした。 The medium is based on YPD medium, using saccharified liquid (potato saccharified liquid) obtained by treating potato pulp with commercial enzyme Acremonium cellulase in Example 1- (2), and sugar beet waste molasses obtained in China Prepared. As a control medium, YPD medium was used. The culture time was 6 hours, 18 hours, or 24 hours.
一般に酵母の培養には廃糖蜜が好適な栄養源として使用されるが、ジャガイモ糖化液を使用することにより、廃糖蜜を上回る酵母の増殖量が得られた（図６）。 In general, molasses is used as a suitable nutrient source for culturing yeast, but by using a potato saccharified solution, the amount of yeast growth exceeding the molasses was obtained (FIG. 6).
この結果は、ジャガイモ塊茎由来原料の糖化物が、炭素源だけでなく、窒素や他の無機成分を含めた総合的な栄養源として優れていることを示している。 This result shows that the saccharified material derived from the potato tuber is excellent not only as a carbon source but also as a comprehensive nutrient source including nitrogen and other inorganic components.
（２）エタノール発酵実験
実施例２−（１）で、凍結乾燥ポテトパルプ（5％）に0.5％、1.5％、又は5％粉末セルロースを添加したものを炭素源として、A. cellulolyticusTN株を培養して得られた酵素液に、酵母Saccharomyces cerevisiae IR-2株を加えて発酵を行った。この発酵においては、酵母を、OD600値が0.1になるように前記糖化液に添加した後、30℃、攪拌速度120 rpmで、２４時間培養した。得られた培養後上清について、エタノール収量を測定した（図７）。エタノールの測定はアミネックスHPX-87H(BioRad 社)カラムを装着した日本分光社製高速液体クロマトグラフィーシステム（検出器：示差屈折計2031Plus）を用いて、行った。
(2) Ethanol fermentation experiment In Example 2- (1), A. cellulolyticus TN strain was cultured using lyophilized potato pulp (5%) added with 0.5%, 1.5%, or 5% powdered cellulose as a carbon source. The yeast Saccharomyces cerevisiae IR-2 strain was added to the enzyme solution thus obtained for fermentation. In this fermentation, yeast was added to the saccharified solution so that the OD 600 value was 0.1, and then cultured at 30 ° C. and a stirring speed of 120 rpm for 24 hours. The ethanol yield of the obtained post-culture supernatant was measured (FIG. 7). The measurement of ethanol was performed using a high-performance liquid chromatography system (detector: differential refractometer 2031Plus) manufactured by JASCO Corporation equipped with an Aminex HPX-87H (BioRad) column.
この結果、糖化液中のグルコースが迅速に消費されてエタノールに変換され、その収量は理論値に対して97%に達した（図７、「ポテトパルプのみ」）。従ってこの方法で得られた糖化液には発酵阻害を起こす物質は含まれないことが示された。 As a result, glucose in the saccharified solution was rapidly consumed and converted into ethanol, and the yield reached 97% of the theoretical value (FIG. 7, “potato pulp only”). Therefore, it was shown that the saccharified solution obtained by this method does not contain substances that cause fermentation inhibition.
本発明の方法は、ジャガイモ塊茎由来原料を酵素分解することで効率的利用を図り、かつそれが廃棄物の場合はその処理を行うために用いることができる。さらに、この糖化により得られる糖を発酵により変換することで、新規な液体燃料の製造も実施することができる。本発明による、ジャガイモ由来原料の分解・糖化技術は、その原料の有効利用、それが廃棄物である場合はその処理に広く適用できる。またこの技術によって得られる糖化物は、液体燃料を効率的に製造するのに有用である。 The method of the present invention can be efficiently used by enzymatic decomposition of a potato tuber-derived raw material, and can be used for the treatment when it is waste. Furthermore, a novel liquid fuel can be produced by converting the sugar obtained by this saccharification by fermentation. The technology for decomposing and saccharifying potato-derived raw materials according to the present invention can be widely applied to the effective use of the raw materials and, when it is waste, the processing thereof. In addition, the saccharified product obtained by this technique is useful for efficiently producing a liquid fuel.
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