JP2011050359A - New microorganism, enzyme derived from the microorganism and method for producing saccharified solution by using the same - Google Patents
New microorganism, enzyme derived from the microorganism and method for producing saccharified solution by using the same Download PDFInfo
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- JP2011050359A JP2011050359A JP2009204940A JP2009204940A JP2011050359A JP 2011050359 A JP2011050359 A JP 2011050359A JP 2009204940 A JP2009204940 A JP 2009204940A JP 2009204940 A JP2009204940 A JP 2009204940A JP 2011050359 A JP2011050359 A JP 2011050359A
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- Prior art keywords
- microorganism
- culture
- enzyme
- lignocellulosic biomass
- saccharified solution
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Images
Classifications
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- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Processing Of Solid Wastes (AREA)
Abstract
Description
本発明は、新規微生物および該微生物由来の酵素、ならびにこれらを用いた糖化液の製造方法に関する。詳しくは、本発明は、リグノセルロース系バイオマスから効率よく糖化液を製造することができる新規微生物および該微生物由来の酵素、ならびにこれらを用いた糖化液の製造方法に関する。 The present invention relates to a novel microorganism, an enzyme derived from the microorganism, and a method for producing a saccharified solution using these. Specifically, the present invention relates to a novel microorganism capable of efficiently producing a saccharified solution from lignocellulosic biomass, an enzyme derived from the microorganism, and a method for producing a saccharified solution using these microorganisms.
産業革命以来、エネルギー資源として、石油、石炭、および天然ガスなどの化石燃料が大量に使用されてきた。その結果、今日、二酸化炭素などの温室効果ガスによる地球温暖化は、もはや一刻の猶予もないほど深刻化している。さらに、これまでの主要なエネルギー資源であった石油の可採年数が数十年との予測がされており、これらの化石燃料に取って代わる、地球に優しい新たなエネルギー資源の確保が急務となっている。 Since the industrial revolution, fossil fuels such as oil, coal, and natural gas have been used in large quantities as energy resources. As a result, global warming due to greenhouse gases such as carbon dioxide is becoming increasingly serious today. Furthermore, it has been predicted that oil, which has been a major energy resource so far, has been available for several decades, and there is an urgent need to secure a new earth-friendly energy resource to replace these fossil fuels. It has become.
これらの問題の一つの解決策として、バイオマス（再生可能な、生物由来の有機性資源で化石資源を除いたもの）から燃料や、生分解性ポリマーなどの原料となる化成品を生産する、バイオリファイナリーの研究・開発が世界各地で進められている。 One solution to these problems is to produce biochemicals that are raw materials such as fuel and biodegradable polymers from biomass (renewable, bio-derived organic resources excluding fossil resources). Refinery research and development is ongoing around the world.
こうしたバイオリファイナリーの中でも、特に、バイオマスエタノール（以下、「バイオエタノール」とも称する）の生産は既に実用化されており、トウモロコシやサトウキビなどの可食部に含まれるデンプン質または糖質を用いたエタノール生産が、米国やブラジルなどで大規模に行われている。しかしながら、これらの食糧資源をバイオエタノールの原料とすることで、食糧価格の高騰や、発展途上国での深刻な食糧不足を引き起こすことが懸念されている。本発明者は、こうした状況を鑑みて、作物の非可食部である葉や茎など（ソフトバイオマス）、あるいは木材など（ハードバイオマス）に含まれるリグノセルロース系バイオマスを原料とするバイオリファイナリー技術の開発に取り組んできた。 Among these biorefinaries, the production of biomass ethanol (hereinafter also referred to as “bioethanol”) has already been put to practical use, and ethanol using starch or sugar contained in edible parts such as corn and sugarcane. Production is carried out on a large scale in the US and Brazil. However, there is a concern that using these food resources as raw materials for bioethanol will cause a rise in food prices and a serious shortage of food in developing countries. In view of these circumstances, the present inventor has developed biorefinery technology using lignocellulosic biomass contained in leaves and stems (soft biomass) or wood (hard biomass) which are non-edible parts of crops as raw materials. I have been working on development.
一般に、バイオリファイナリーのプロセスは、（ｉ）バイオマスに含まれる多糖類を糖化（加水分解）することによって単糖類を含む糖化液を得る工程と、（ｉｉ）得られた糖化液に含まれる単糖類に、微生物または酵素を作用させることによって各種の有用化合物に変換する工程との２つの工程を含む。しかしながら、作物の葉や茎または木材のようなリグノセルロース系バイオマスは、構造性多糖であるセルロースに芳香族化合物の重合体であるリグニンおよびヘミセルロースが強固に結合する構造を有しており、デンプン質や糖質を主成分とするデンプン系バイオマスや糖質系バイオマスと比較して、加水分解反応の効率が著しく低いことが知られている。そこで、リグノセルロース系バイオマスを高効率で糖化するための前処理または加水分解反応に関する技術開発が進められている。 In general, the biorefinery process includes (i) a step of obtaining a saccharified solution containing monosaccharides by saccharifying (hydrolyzing) polysaccharides contained in biomass, and (ii) monosaccharides contained in the obtained saccharified solution. In addition, there are two steps including conversion to various useful compounds by the action of microorganisms or enzymes. However, lignocellulosic biomass, such as crop leaves, stems, or wood, has a structure in which lignin and hemicellulose, which are polymers of aromatic compounds, are tightly bound to cellulose, which is a structural polysaccharide. It is known that the efficiency of the hydrolysis reaction is remarkably low compared with starch-based biomass and saccharide-based biomass whose main components are sugar and sugar. Therefore, technological development relating to pretreatment or hydrolysis reaction for saccharifying lignocellulosic biomass with high efficiency is in progress.
リグノセルロース系バイオマスの加水分解法は、主に酸または塩基を用いる化学的加水分解法と、微生物等が生産する酵素（セルラーゼ）を用いる生物学的加水分解法の２種に大別される。後者の生物学的加水分解法によると、温和な条件で、発酵阻害物質が少ない高濃度の糖化液を得ることができる。そこで、より高活性なセルラーゼを産生する微生物の探索が進められている。 Lignocellulosic biomass hydrolysis methods are roughly classified into two types: chemical hydrolysis methods mainly using acids or bases, and biological hydrolysis methods using enzymes (cellulases) produced by microorganisms and the like. According to the latter biological hydrolysis method, a high-concentration saccharified solution with a small amount of fermentation-inhibiting substances can be obtained under mild conditions. Therefore, search for microorganisms that produce more highly active cellulase is underway.
例えば、特許文献１ではリグノセルロース系バイオマスに含まれる結晶性セルロースに対して高い加水分解活性を有するトリコデルマ・リーゼイ（Ｔｒｉｃｈｏｄｅｒｍａ ｒｅｅｓｅｉ）および該微生物の製造方法が開示されている。 For example, Patent Document 1 discloses Trichoderma reesei having high hydrolytic activity for crystalline cellulose contained in lignocellulosic biomass and a method for producing the microorganism.
しかしながら、上記特許文献１に記載のトリコデルマ・リーゼイを用いても、糖化効率が未だ不十分であり、さらなる改良が望まれているのが現状である。 However, even if Trichoderma reesei described in Patent Document 1 is used, the saccharification efficiency is still insufficient, and further improvement is desired.
そこで本発明は、高効率で糖化液を得るための手段を提供することを目的とする。 Accordingly, an object of the present invention is to provide a means for obtaining a saccharified solution with high efficiency.
本発明者は、上記の問題を解決すべく、鋭意研究を行った。その結果、リグノセルロース系バイオマス分解能を有するペニシリウム属（Ｐｅｎｉｃｉｌｌｉｕｍ ｓｐ．）に属する微生物を用いることによってリグノセルロース系バイオマスから高効率で糖化液を製造できることを見出し、本発明を完成させた。 The present inventor has intensively studied to solve the above problems. As a result, the present inventors have found that a saccharified solution can be produced from lignocellulosic biomass with high efficiency by using a microorganism belonging to the genus Penicillium sp. Having lignocellulosic biomass resolving power, and the present invention has been completed.
すなわち、本発明は、リグノセルロース系バイオマス分解能を有するペニシリウム属（Ｐｅｎｉｃｉｌｌｉｕｍ ｓｐ．）に属する微生物である。 That is, the present invention is a microorganism belonging to the genus Penicillium sp. Having a lignocellulosic biomass resolving power.
本発明によれば、リグノセルロース系バイオマスから高効率で糖化液を製造することができる。 According to the present invention, a saccharified solution can be produced from lignocellulosic biomass with high efficiency.
以下、本発明の好ましい形態を説明する。なお、本発明の技術的範囲は特許請求の範囲の記載に基づいて定められるべきであり、以下の形態のみに制限されない。 Hereinafter, preferred embodiments of the present invention will be described. The technical scope of the present invention should be determined based on the description of the scope of claims, and is not limited to the following embodiments.
＜微生物＞
本形態の微生物は、ペニシリウム属（Ｐｅｎｉｃｉｌｌｉｕｍ ｓｐ．）に属し、リグノセルロース系バイオマス分解能を有する微生物である。該微生物はリグノセルロース系バイオマスの分解活性に優れるため、リグノセルロース系バイオマスから高効率で糖化液を製造することができる。このようなペニシリウム属に属する微生物のうち、本発明者によって新たに見出されたペニシリウム Ｓ１１Ｄ６６０８−Ｍ１（受託番号：ＦＥＲＭ ＢＰ−１１０８３）および／またはペニシリウム Ｓ１１Ｄ６６０８−Ｍ２（受託番号：ＦＥＲＭ ＢＰ−１１０８４）であることが好ましい。なお、本明細書において、ペニシリウム Ｓ１１Ｄ６６０８−Ｍ１を略して「菌株Ｍ１」と、ペニシリウム Ｓ１１Ｄ６６０８−Ｍ２を略して「菌株Ｍ２」と、それぞれ称する場合がある。以下で、菌株Ｍ１および菌株Ｍ２について説明する。
<Microorganism>
The microorganism of this form belongs to the genus Penicillium sp. And has a lignocellulosic biomass resolving power. Since the microorganism is excellent in the decomposition activity of lignocellulosic biomass, a saccharified solution can be produced from the lignocellulosic biomass with high efficiency. Among the microorganisms belonging to the genus Penicillium, Penicillium S11D6608-M1 (Accession Number: FERM BP-11083) and / or Penicillium S11D6608-M2 (Accession Number: FERM BP-11084) newly found by the present inventors It is preferable that In the present specification, Penicillium S11D6608-M1 may be abbreviated as “strain M1” and Penicillium S11D6608-M2 may be abbreviated as “strain M2”. Hereinafter, the strain M1 and the strain M2 will be described.
［１次スクリーニング］
中国東北部で生産されたトウモロコシのコーンコブを入手した。該コーンコブの表面の一部にはカビが観察された。該コーンコブを室温で約２ヵ月間風乾し、金切ばさみで１〜５ｃｍ程度に切断した。そして、これを家庭用ミキサーに５分間かけることにより粗粉砕した。該粗粉砕物をさらにボールミル（ＡＮＳ−６０Ｓ、日陶科学社製）を用いて微粉化した。得られたコーンコブ粉砕物の粒径分布を下記表１に示す。なお、本明細書において「粒径」とは、粒子の輪郭線上の任意の２点間の距離のうち、最大の距離を意味する。また、粒径の値は、試験用ふるいによって測定した値を採用する。下記表１の各粒径における粉砕物の割合の求め方は以下のとおりである。まず、３種類の試験用ふるい（呼び寸法０．５ｍｍ、１．０ｍｍ、１．７ｍｍ；ＳＡＮＰＯ社製）を呼び寸法が小さいものが下になるように順次重ね、最上部のふるいに上記粉砕物５ｇを入れた。これを間欠的に５分間ふるい、それぞれのふるいに残存した粉砕物の質量を測定し、それぞれの割合を求めた。
[Primary screening]
We obtained corn cob produced in northeastern China. Mold was observed on a part of the surface of the corn cob. The corn cob was air-dried at room temperature for about 2 months, and cut into about 1 to 5 cm with a gold cutter. And this was coarsely pulverized by applying to a home mixer for 5 minutes. The coarsely pulverized product was further pulverized using a ball mill (ANS-60S, manufactured by Nichido Kagaku Co., Ltd.). Table 1 shows the particle size distribution of the pulverized corn cob obtained. In the present specification, the “particle diameter” means the maximum distance among the distances between any two points on the particle outline. Moreover, the value measured with the sieve for a test is employ | adopted for the value of a particle size. The method of obtaining the ratio of the pulverized product at each particle size shown in Table 1 below is as follows. First, three types of test sieves (nominal dimensions 0.5 mm, 1.0 mm, 1.7 mm; manufactured by SANPO) are stacked one on top of the other so that the smaller nominal dimensions are on the bottom. 5 g was added. This was sieved intermittently for 5 minutes, the mass of the pulverized material remaining on each sieve was measured, and the respective proportions were determined.
該コーンコブ粉砕物のうち、粒径が１．０ｍｍ未満の粉砕物１ｇを試験管に取り、滅菌水道水１０ｍｌを加えて懸濁し、恒温振盪培養装置を用いて３２℃、回転数２２０ｐｐｍで７日間振盪培養した。培養後の各試験管中の培養液を一部サンプリングし、培養液中に含まれる全糖量をフェノール硫酸法により分析して、全糖量が特に多かった培養液を選定した。全糖量が多かった培養液では、コーンコブに付着した微生物が、コーンコブに含まれるリグノセルロースを効率よく糖化したものと考えられた。なお、本明細書において、フェノール硫酸法による分析は、中村道徳編、貝沼圭二編、「生物化学実験法（２５）澱粉・関連糖質酵素実験法」、学会出版センター版、１９８９年１０月発行、２０６頁に記載の方法に準じて行った。 Of the corn cob pulverized product, 1 g of pulverized product having a particle size of less than 1.0 mm is placed in a test tube, suspended by adding 10 ml of sterilized tap water, and using a constant temperature shaking culture apparatus at 32 ° C. and a rotation speed of 220 ppm for 7 days. Cultured with shaking. A part of the culture solution in each test tube after the culture was sampled, and the total amount of sugar contained in the culture solution was analyzed by the phenol-sulfuric acid method to select a culture solution having a particularly high total amount of sugar. In the culture solution with a large amount of total sugar, it was considered that microorganisms attached to corn cob efficiently saccharified lignocellulose contained in corn cob. In this specification, the analysis by the phenol-sulfuric acid method is performed by Michinori Nakamura, Junji Kakinuma, “Biochemical Experimental Method (25) Starch / Related Carbohydrate Enzyme Experimental Method”, Academic Publishing Center Edition, published in October 1989. , Page 206.
［１次スクリーニングした菌の単胞子分離］
上記で選定した培養液を滅菌水で適当に希釈し、下記表２に示す組成のＰＤＹ寒天培地（オートクレーブにて１２１℃、２０分間滅菌）に塗布し、３２℃のインキュベーター中でプレート培養した。培養開始から１４日後に培地上にコロニーの形成が認められた。このうち、明らかにカビ以外の微生物であると思われるコロニーを除外し、カビ状でかつ形状が大きなコロニーを選別した。
[Single-spore isolation of primary screened bacteria]
The culture solution selected above was appropriately diluted with sterilized water, applied to a PDY agar medium (sterilized in an autoclave at 121 ° C. for 20 minutes) having the composition shown in Table 2 below, and cultured in a plate at 32 ° C. in an incubator. Colony formation was observed on the medium 14 days after the start of the culture. Among these, colonies that were apparently microorganisms other than mold were excluded, and moldy colonies having a large shape were selected.
［２次スクリーニング］
（前培養）
２００ｍｌ三角フラスコに下記表３に示す組成のＡ培地２０ｍｌを入れ、オートクレーブで１２１℃、２０分間滅菌した。該Ａ培地に上記１次スクリーニングで選定した菌株を１白金耳接種した。これを、恒温振盪培養装置を用いて３２℃、回転数２２０ｐｐｍで２４時間振盪培養した。
[Secondary screening]
(Pre-culture)
In a 200 ml Erlenmeyer flask, 20 ml of medium A having the composition shown in Table 3 below was placed, and sterilized by autoclaving at 121 ° C. for 20 minutes. One platinum loop of the strain selected in the primary screening was inoculated into the A medium. This was subjected to shaking culture at 32 ° C. and a rotation speed of 220 ppm using a constant temperature shaking culture apparatus for 24 hours.
次に、２００ｍｌ三角フラスコに下記表４に示す組成のＢ培地２０ｍｌを入れ、オートクレーブで１２１℃、２０分間滅菌した。該Ｂ培地に、上記で培養した前培養液を５（ｖ／ｖ）％植菌した。これを、恒温振盪培養装置を用いて３２℃、回転数２２０ｐｐｍで６日間振盪培養した。 Next, 20 ml of the B medium having the composition shown in Table 4 below was placed in a 200 ml Erlenmeyer flask and sterilized by autoclaving at 121 ° C. for 20 minutes. The B medium was inoculated with 5% (v / v)% of the precultured medium cultured above. This was subjected to shaking culture at 32 ° C. and a rotation speed of 220 ppm for 6 days using a constant temperature shaking culture apparatus.
（本培養）
上記１次スクリーニングで調製した粒径が１．０ｍｍ未満のコーンコブ粉砕物０．２ｇを、上記培養液のろ液を適当に水道水で希釈した希釈培養液２０ｍｌに懸濁し、５０℃で３日間糖化を行った。得られた糖化液中に含まれる全糖量をフェノール硫酸法により分析し、全糖量が多かった糖化液に対応した２菌株を、リグノセルロース系バイオマスの糖化力に優れている菌株として選定した。これらをそれぞれ菌株Ｉおよび菌株ＩＩと称した。
(Main culture)
0.2 g of a corn cob pulverized product having a particle size of less than 1.0 mm prepared in the primary screening is suspended in 20 ml of a diluted culture solution obtained by appropriately diluting the filtrate of the culture solution with tap water, and the mixture is kept at 50 ° C. for 3 days. Saccharification was performed. The total amount of sugar contained in the obtained saccharified solution was analyzed by the phenol-sulfuric acid method, and two strains corresponding to the saccharified solution having a large total amount of sugar were selected as strains having excellent saccharifying power of lignocellulosic biomass. . These were designated as strain I and strain II, respectively.
［分類学的性質］
上記でスクリーニングした菌株Ｉおよび菌株ＩＩの分類学的性質を以下で述べる。
[Taxonomic properties]
The taxonomic properties of strains I and II screened above are described below.
（１）菌株Ｉ
（ａ）形態的性質
以下の（ｉ）〜（ｉｖ）培地において、２５℃で３週間培養後のコロニーの形態を観察した。なお、以下の表５〜８において、色調の括弧内の英数文字は「Ｋｏｒｎｅｒｕｐ ａｎｄ Ｗａｎｓｃｈｅｒ（１９７８）」で用いられている色のコード ナンバーを表す。
(1) Strain I
(A) Morphological properties In the following media (i) to (iv), the morphology of colonies after culturing at 25 ° C. for 3 weeks was observed. In Tables 5 to 8 below, the alphanumeric characters in the parentheses of the color tone represent the code numbers of the colors used in “Kornerup and Wancher (1978)”.
（ｉ）ポテトデキストロース寒天培地（商品名：ダイゴ、日本製薬社製） (I) Potato dextrose agar medium (trade name: Daigo, manufactured by Nippon Pharmaceutical Co., Ltd.)
（ｉｉ）２％ 麦芽寒天培地（Ｍａｌｔ Ａｇａｒ） (Ii) 2% malt agar medium (Malt Agar)
（ｉｉｉ）バクトオートミール寒天培地（Ｂａｃｔｏ Ｏａｔｍｅａｌ Ａｇａｒ、ベクトン ディッキンソン社製） (Iii) Bacto oatmeal agar (Bacto Oatmeal Agar, manufactured by Becton Dickinson)
（ｉｖ）ＬＣＡ（三浦培地） (Iv) LCA (Miura medium)
その他の微視的観察結果を表９に示す。 Table 9 shows other microscopic observation results.
（ｂ）同定
アポロンＤＢ−ＦＵに対するＢＬＡＳＴ（Ａｌｔｓｃｈｕｌ ｅｔ ａｌ．，１９９７）相同性検索の結果、菌株Ｉの２８Ｓ ｒＤＮＡ−Ｄ１／Ｄ２塩基配列は、子嚢菌類の一種であるＰｅｎｉｃｉｌｌｉｕｍ ａｃｕｌｅａｔｕｍの塩基配列と９９．５％の相同率を示した。ＧｅｎＢａｎｋ／ＤＤＢＪ／ＥＭＢＬなどの国際塩基配列データベースに対する相同性検索の結果においては、菌株Ｉの２８Ｓ ｒＤＮＡ−Ｄ１／Ｄ２塩基配列は子嚢菌類の一種であるＰｅｎｉｃｉｌｌｉｕｍ ｃｆ． ｖｅｒｒｕｃｕｌｏｓｕｍ ＲＳ７ＰＦ（この菌種名の「ｃｆ．」は、Ｐ． ｖｅｒｒｕｃｕｌｏｓｕｍに類似する菌株であることを表す）、Ｐ． ａｃｕｌｅａｔｕｍ、Ｐ． ｖｅｒｒｕｃｕｌｏｓｕｍの塩基配列と高い相同率を示した。アポロンＤＢ−ＦＵおよび国際塩基配列データベースに対する相同性検索で得られた上位の塩基配列をもとに作成した系統樹において、菌株ＩはＰｅｎｉｃｉｌｌｉｍ ｃｆ． ｖｅｒｒｕｃｕｌｏｓｕｍ ＲＳ７ＰＦと同一の系統枝を形成した。
(B) Identification As a result of BLAST (Altschul et al., 1997) homology search for apolone DB-FU, the 28S rDNA-D1 / D2 base sequence of strain I is the base sequence of Penicillium aculeatum, which is a kind of Ascomycetes. A homology rate of 99.5% was shown. As a result of homology search against an international nucleotide sequence database such as GenBank / DDBJ / EMBL, the 28S rDNA-D1 / D2 nucleotide sequence of strain I is Penicillium cf. verruculosum RS7PF ("cf." in this bacterial species name represents a strain similar to P. verruculosum), P. verruculosum RS7PF. aculeatum, P.A. It showed high homology with the base sequence of verruculosum. In the phylogenetic tree created based on the higher order base sequences obtained by homology search against Apollon DB-FU and the international base sequence database, the strain I is Penicillim cf. The same branch was formed as verruculosum RS7PF.
菌株ＩのＩＴＳ−５．８Ｓ ｒＤＮＡ塩基配列は、アポロンＤＢ−ＦＵに対するＢＬＡＳＴ相同性検索の結果、子嚢菌類の一種である、Ｐ． ｖｅｒｒｕｃｕｌｏｓｕｍ の塩基配列と９８．８％の相同率を示した。また、ＧｅｎＢａｎｋ／ＤＤＢＪ／ＥＭＢＬなどの国際塩基配列データベースに対する相同性検索の結果においては、菌株ＩのＩＴＳ−５．８Ｓ ｒＤＮＡ塩基配列は、Ｐ． ｐｉｎｏｐｈｉｌｕｍの塩基配列と１００％の相同率を示した。国際塩基配列データベースおよびアポロンＤＢ−ＦＵに対する相同性検索で得られた上位の塩基配列をもとに作成した系統樹において、菌株ＩはＰ． ｐｉｎｏｐｈｉｌｕｍと同一の系統枝を形成した。 The ITS-5.8S rDNA base sequence of strain I is a kind of ascomycetous fungi as a result of BLAST homology search against apolone DB-FU. It showed 98.8% homology with the base sequence of verruculosum. Moreover, in the result of the homology search with respect to the international base sequence database such as GenBank / DDBJ / EMBL, the ITS-5.8S rDNA base sequence of strain I is P.P. It showed 100% homology with the base sequence of pinophilum. In the phylogenetic tree created based on the high-order base sequences obtained by the homology search for the international base sequence database and Apollon DB-FU, The same phylogenetic branch as that of pinophilum was formed.
したがって、２８Ｓ ｒＤＮＡ−Ｄ１／Ｄ２塩基配列の解析結果から、菌株ＩはＰｅｎｉｃｉｌｌｉｍ ｃｆ． ｖｅｒｒｕｃｕｌｏｓｕｍに近縁であるものの、ＩＴＳ−５．８Ｓ ｒＤＮＡ塩基配列の解析で本検体と同一系統枝を形成した２８Ｓ ｒＤＮＡ−Ｄ１／Ｄ２塩基配列のデータが登録されてないことから、本検体の種レベルの推定は困難であると思われた。よって、菌株ＩはＰ． ｐｉｎｏｐｈｉｌｕｍに近縁なＰｅｎｉｃｉｌｌｉｕｍ属の一種であると考えられた。なお、Ｐ． ｖｅｒｒｕｃｕｌｏｓｕｍとＰ． ｐｉｎｏｐｈｉｌｕｍとは、分類学的に非常に近縁であることが知られている。 Therefore, from the analysis result of 28S rDNA-D1 / D2 nucleotide sequence, strain I was found to be Penicillim cf. Although it is closely related to verruculosum, the 28S rDNA-D1 / D2 nucleotide sequence data that formed the same phylogeny with this sample in the analysis of the ITS-5.8S rDNA nucleotide sequence has not been registered. The level estimation seemed difficult. Therefore, strain I is P. It was considered to be a kind of Penicillium genus closely related to pinophilum. P.P. verruculosum and P. et al. It is known that pinophyllum is very close taxonomically.
コロニー性状および形態観察の結果、菌株Ｉは赤褐色〜茶褐色系のコロニーを形成し、培地中に顕著な赤色系の可溶性色素を産出した。また、有隔壁の栄養菌糸のみの形成が観察された。今回の観察結果では、胞子などの生殖器官の形成が観察されなかったことから、分類群の推定はできなかった。なお、ＩＴＳ−５．８Ｓ ｒＤＮＡ塩基配列解析結果より菌株Ｉと近縁を推定されるＰ． ｐｉｎｏｐｈｉｌｕｍはまれに赤色系の色素を産出することが知られている。 As a result of colony properties and morphological observation, the strain I formed a reddish brown to brownish brown colony and produced a remarkable reddish soluble pigment in the medium. In addition, only the formation of vegetative mycelium in the septum was observed. In this observation, no taxonomic group could be estimated because the formation of reproductive organs such as spores was not observed. It should be noted that P. aphisae strains closely related to strain I are estimated from the results of ITS-5.8S rDNA nucleotide sequence analysis. Pinophilum is known to rarely produce red pigments.
以上の２８Ｓ ｒＤＮＡ−Ｄ１／Ｄ２およびＩＴＳ−５．８Ｓ ｒＤＮＡ塩基配列解析、ならびにコロニー性状および形態観察の結果から総合的に鑑みて、菌株ＩはＰ． ｐｉｎｏｐｈｉｌｕｍに近縁なＰｅｎｉｃｉｌｌｉｕｍ属に帰属する新規な菌であると判断し、ペニシリウム Ｓ１１Ｄ６６０８−Ｍ１（Ｐｅｎｉｃｉｌｌｉｕｍ ｓｐ． Ｓ１１Ｄ６６０８−Ｍ１）と命名した。 In view of the above results of 28S rDNA-D1 / D2 and ITS-5.8S rDNA nucleotide sequence analysis and colony properties and morphology observation, strain I is P. It was judged to be a novel bacterium belonging to the genus Penicillium that is closely related to pinophyllum, and was named Penicillium S11D6608-M1 (Penicillium sp. S11D6608-M1).
なお、本菌株は、２００８年１２月２４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に受託番号ＦＥＲＭ ＢＰ−１１０８３として、Ｓ１１Ｄ６６０８−Ｍ１の名称で寄託されている。 This strain has been deposited under the name of S11D6608-M1 under the accession number FERM BP-11083 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center on December 24, 2008.
（２）菌株ＩＩ
（ａ）形態的性質
以下の（ｉ）〜（ｉｖ）培地において、２５℃で３週間培養後のコロニーの形態を観察した。なお、以下の表１０〜１３において、色調の括弧内の英数文字は「Ｋｏｒｎｅｒｕｐ ａｎｄ Ｗａｎｓｃｈｅｒ（１９７８）」で用いられている色のコード ナンバーを表す。
(2) Strain II
(A) Morphological properties In the following media (i) to (iv), the morphology of colonies after culturing at 25 ° C. for 3 weeks was observed. In Tables 10 to 13 below, the alphanumeric characters in the parentheses of the color tone represent the code numbers of the colors used in “Kornerup and Wancher (1978)”.
（ｉ）ポテトデキストロース寒天培地（商品名：ダイゴ、日本製薬社製） (I) Potato dextrose agar medium (trade name: Daigo, manufactured by Nippon Pharmaceutical Co., Ltd.)
（ｉｉ）２％ 麦芽寒天培地（Ｍａｌｔ Ａｇａｒ） (Ii) 2% malt agar medium (Malt Agar)
（ｉｉｉ）バクトオートミール寒天培地（Ｂａｃｔｏ Ｏａｔｍｅａｌ Ａｇａｒ、ベクトン ディッキンソン社製） (Iii) Bacto oatmeal agar (Bacto Oatmeal Agar, manufactured by Becton Dickinson)
（ｉｖ）ＬＣＡ（三浦培地） (Iv) LCA (Miura medium)
その他の微視的観察結果を表１４に示す。 Table 14 shows other microscopic observation results.
（ｂ）同定
アポロンＤＢ−ＦＵに対するＢＬＡＳＴ（Ａｌｔｓｃｈｕｌ ｅｔ ａｌ．，１９９７）相同性検索の結果、菌株ＩＩの２８Ｓ ｒＤＮＡ−Ｄ１／Ｄ２塩基配列は、子嚢菌類の一種であるＰｅｎｉｃｉｌｌｉｕｍ ａｃｕｌｅａｔｕｍの塩基配列と９９．５％の相同率を示した。ＧｅｎＢａｎｋ／ＤＤＢＪ／ＥＭＢＬなどの国際塩基配列データベースに対する相同性検索の結果においては、菌株ＩＩの２８Ｓ ｒＤＮＡ−Ｄ１／Ｄ２塩基配列は子嚢菌類の一種であるＰｅｎｉｃｉｌｌｉｕｍ ｃｆ． ｖｅｒｒｕｃｕｌｏｓｕｍ ＲＳ７ＰＦ（この菌種名の「ｃｆ．」は、Ｐ． ｖｅｒｒｕｃｕｌｏｓｕｍに類似する菌株であることを表す）、Ｐ． ａｃｕｌｅａｔｕｍ、Ｐ． ｖｅｒｒｕｃｕｌｏｓｕｍの塩基配列と高い相同率を示した。アポロンＤＢ−ＦＵおよび国際塩基配列データベースに対する相同性検索で得られた上位の塩基配列をもとに作成した系統樹において、菌株ＩＩはＰｅｎｉｃｉｌｌｉｍ ｃｆ． ｖｅｒｒｕｃｕｌｏｓｕｍ ＲＳ７ＰＦと同一の系統枝を形成した。
(B) Identification As a result of BLAST (Altschul et al., 1997) homology search for apolone DB-FU, the 28S rDNA-D1 / D2 base sequence of strain II is the base sequence of Penicillium aculeatum, which is a kind of Ascomycetes A homology rate of 99.5% was shown. As a result of homology search against an international nucleotide sequence database such as GenBank / DDBJ / EMBL, the 28S rDNA-D1 / D2 nucleotide sequence of strain II is Penicillium cf. verruculosum RS7PF ("cf." in this bacterial species name represents a strain similar to P. verruculosum), P. verruculosum RS7PF. aculeatum, P.A. It showed high homology with the base sequence of verruculosum. In the phylogenetic tree created based on the higher order base sequences obtained by homology search against Apollon DB-FU and the international base sequence database, the strain II is Penicillim cf. The same branch was formed as verruculosum RS7PF.
菌株ＩＩのＩＴＳ−５．８Ｓ ｒＤＮＡ塩基配列は、アポロンＤＢ−ＦＵに対するＢＬＡＳＴ相同性検索の結果、子嚢菌類の一種である、Ｐ． ｖｅｒｒｕｃｕｌｏｓｕｍ の塩基配列と９８．８％の相同率を示した。また、ＧｅｎＢａｎｋ／ＤＤＢＪ／ＥＭＢＬなどの国際塩基配列データベースに対する相同性検索の結果においては、菌株ＩＩのＩＴＳ−５．８Ｓ ｒＤＮＡ塩基配列は、Ｐ． ｐｉｎｏｐｈｉｌｕｍの塩基配列と１００％の相同率を示した。国際塩基配列データベースおよびアポロンＤＢ−ＦＵに対する相同性検索で得られた上位の塩基配列をもとに作成した系統樹において、菌株ＩＩはＰ． ｐｉｎｏｐｈｉｌｕｍと同一の系統枝を形成した。 The ITS-5.8S rDNA base sequence of strain II is a kind of ascomycetous fungi as a result of BLAST homology search against apolone DB-FU. It showed 98.8% homology with the base sequence of verruculosum. Moreover, in the result of the homology search with respect to international base sequence databases such as GenBank / DDBJ / EMBL, the ITS-5.8S rDNA base sequence of strain II is P.P. It showed 100% homology with the base sequence of pinophilum. In the phylogenetic tree created based on the high-order base sequences obtained by the homology search for the international base sequence database and Apollon DB-FU, The same phylogenetic branch as that of pinophilum was formed.
したがって、２８Ｓ ｒＤＮＡ−Ｄ１／Ｄ２塩基配列の解析結果から、菌株ＩＩはＰｅｎｉｃｉｌｌｉｍ ｃｆ． ｖｅｒｒｕｃｕｌｏｓｕｍに近縁であるものの、ＩＴＳ−５．８Ｓ ｒＤＮＡ塩基配列の解析で本検体と同一系統枝を形成した２８Ｓ ｒＤＮＡ−Ｄ１／Ｄ２塩基配列のデータが登録されてないことから、本検体の種レベルの推定は困難であると思われた。よって、菌株ＩＩはＰ． ｐｉｎｏｐｈｉｌｕｍに近縁なＰｅｎｉｃｉｌｌｉｕｍ属の一種であると考えられた。なお、Ｐ． ｖｅｒｒｕｃｕｌｏｓｕｍとＰ． ｐｉｎｏｐｈｉｌｕｍとは、分類学的に非常に近縁であることが知られている。 Therefore, from the analysis result of 28S rDNA-D1 / D2 nucleotide sequence, strain II was found to be Penicillim cf. Although it is closely related to verruculosum, the 28S rDNA-D1 / D2 nucleotide sequence data that formed the same phylogeny with this sample in the analysis of the ITS-5.8S rDNA nucleotide sequence has not been registered. The level estimation seemed difficult. Therefore, strain II is P. It was considered to be a kind of Penicillium genus closely related to pinophilum. P.P. verruculosum and P. et al. It is known that pinophyllum is very close taxonomically.
コロニー性状および形態観察の結果、菌株ＩＩは白色〜赤黄色系のコロニーを形成し、有隔壁の栄養菌糸のみの形成が観察された。今回の観察結果では、胞子などの生殖器官の形成が観察されなかったことから、分類群の推定はできなかった。上述の菌株Ｉのコロニー性状とは顕著な違いが認められたが、菌糸の形状において差異は認められなかった。 As a result of colony properties and morphology observation, the strain II formed white to red-yellow colonies, and formation of only vegetative mycelium with a partition wall was observed. In this observation, no taxonomic group could be estimated because the formation of reproductive organs such as spores was not observed. Although a significant difference was observed from the colony characteristics of the strain I described above, no difference was observed in the shape of the mycelium.
以上の２８Ｓ ｒＤＮＡ−Ｄ１／Ｄ２およびＩＴＳ−５．８Ｓ ｒＤＮＡ塩基配列解析、ならびにコロニー性状および形態観察の結果から総合的に鑑みて、菌株ＩはＰ． ｐｉｎｏｐｈｉｌｕｍに近縁なＰｅｎｉｃｉｌｌｉｕｍ属に帰属する新規な菌であると判断し、ペニシリウム Ｓ１１Ｄ６６０８−Ｍ２（Ｐｅｎｉｃｉｌｌｉｕｍ ｓｐ． Ｓ１１Ｄ６６０８−Ｍ２）と命名した。 In view of the above results of 28S rDNA-D1 / D2 and ITS-5.8S rDNA nucleotide sequence analysis and colony properties and morphology observation, strain I is P. It was judged to be a novel bacterium belonging to the genus Penicillium closely related to pinophilum, and named Penicillium S11D6608-M2 (Penicillium sp. S11D6608-M2).
なお、本菌株は、２００８年１２月２４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に受託番号ＦＥＲＭ ＢＰ−１１０８４として、Ｓ１１Ｄ６６０８−Ｍ２の名称で寄託されている。 This strain was deposited under the name of S11D6608-M2 under the accession number FERM BP-11084 at the National Institute of Advanced Industrial Science and Technology (AIST) on December 24, 2008.
また、本発明の微生物として、より好ましくは、より高効率で糖化液を得られるよう、菌株Ｍ１または菌株Ｍ２に公知の変異処理を施すことによって得られた変異株であることが好ましい。変異処理の方法は、特に制限はないが、例えば、紫外線照射やニトロソグアニジンなどの変異誘発剤を使用することによって変異誘導処理する方法が挙げられる。そして、変異処理を施した菌株から、リグノセルロース系バイオマスの分解活性に優れる菌株を選定する。 The microorganism of the present invention is more preferably a mutant obtained by subjecting strain M1 or strain M2 to a known mutation treatment so that a saccharified solution can be obtained with higher efficiency. The method for the mutation treatment is not particularly limited, and examples thereof include a method for mutagenesis treatment by using a mutagen such as ultraviolet irradiation or nitrosoguanidine. And the strain which is excellent in the decomposition activity of lignocellulosic biomass is selected from the strain which performed the mutation treatment.
＜酵素＞
本発明の他の形態としては、上述の微生物に由来する酵素が挙げられる。該酵素は、リグノセルロース系バイオマスに対して優れた加水分解活性能を有するので、該酵素をリグノセルロース系バイオマスに作用させることにより高効率で糖化液を製造することができる。
<Enzyme>
Other forms of the present invention include enzymes derived from the above-mentioned microorganisms. Since the enzyme has an excellent hydrolytic activity for lignocellulosic biomass, a saccharified solution can be produced with high efficiency by allowing the enzyme to act on lignocellulosic biomass.
本形態の酵素は、上述の微生物の培養物から、従来公知の酵素採取手段および精製手段を用いて調製することができる。酵素採取法としては、例えば、微生物を含む培養物を遠心分離またはろ過することによって、菌体とろ液とを分離する。そして、該菌体およびろ液から、塩析法、等電点沈殿法、溶媒沈殿法（メタノール、エタノール、アセトン、およびイソプロパノールなど）によりタンパク質である酵素を沈殿させたり、限外ろ過により濃縮したりする方法が挙げられる。 The enzyme of this embodiment can be prepared from the above-mentioned microorganism culture by using conventionally known enzyme collecting means and purification means. As an enzyme collection method, for example, a bacterial cell and a filtrate are separated by centrifuging or filtering a culture containing microorganisms. Then, the enzyme, which is a protein, is precipitated from the cells and the filtrate by salting out, isoelectric point precipitation, solvent precipitation (such as methanol, ethanol, acetone, and isopropanol), or concentrated by ultrafiltration. The method to do is mentioned.
リグノセルロース系バイオマスの加水分解には、上記の方法によって得られる粗酵素を用いてもよいが、必要によりさらに精製した酵素を用いることもできる。酵素の精製法としては、例えば、吸着クロマトグラフィー、イオン交換クロマトグラフィー、およびゲルクロマトグラフィーなどのクロマトグラフィーを単独で、または２種以上を組み合わせた精製法が挙げられる。 For hydrolysis of lignocellulosic biomass, a crude enzyme obtained by the above method may be used, but a purified enzyme may be used as necessary. Examples of the enzyme purification method include purification methods such as adsorption chromatography, ion exchange chromatography, and gel chromatography alone or in combination of two or more.
＜糖化液の製造方法＞
本発明の他の形態にかかる糖化液の製造方法は、上述の本発明の微生物をリグノセルロース系バイオマスを含む培地中で培養する工程、または上述の本発明の酵素をリグノセルロース系バイオマスに作用させることにより加水分解する工程を含む。
<Method for producing saccharified solution>
The method for producing a saccharified solution according to another aspect of the present invention includes a step of culturing the above-described microorganism of the present invention in a medium containing lignocellulosic biomass, or causing the above-described enzyme of the present invention to act on lignocellulosic biomass. The process of hydrolyzing by this.
本形態の糖化液の製造方法で用いるリグノセルロース系バイオマスは、セルロースおよびヘミセルロース、ならびにリグニンを含む有機資源であれば植物性でも動物性でも特に制限はなく、農作物、草木類、および海藻類、ならびにこれらの処理物または廃棄物などが含まれる。より具体的には、稲わら、籾殻、麦わら、ふすま、バガス、ヤシ殻、コーンコブ、コットン、ラミー、ケナフ、ビート、大豆粕、コーヒー粕、雑草、木材、竹、バクテリアセルロース、パルプ、紙、セルロース粉末、結晶セルロース、レーヨン、アルカリセルロース、リン酸膨潤セルロース、およびカルボキシメチルセルロースなどが挙げられる。なお、本発明の微生物または酵素は、上記リグノセルロース系バイオマスの糖化能に優れるだけでなく、でんぷん系バイオマスまたは糖質系バイオマスに対しても優れた糖化能を示す。 The lignocellulosic biomass used in the method for producing a saccharified liquid of this embodiment is not particularly limited in terms of plants or animals as long as it is an organic resource containing cellulose and hemicellulose, and lignin. Agricultural crops, plants and seaweeds, and These treated products or wastes are included. More specifically, rice straw, rice husk, wheat straw, bran, bagasse, coconut husk, corn cob, cotton, ramie, kenaf, beet, soybean meal, coffee pod, weed, wood, bamboo, bacterial cellulose, pulp, paper, cellulose Examples thereof include powder, crystalline cellulose, rayon, alkali cellulose, phosphoric acid swollen cellulose, and carboxymethyl cellulose. The microorganism or enzyme of the present invention not only has excellent saccharification ability of the above lignocellulosic biomass, but also exhibits excellent saccharification ability for starch-based biomass or saccharide-based biomass.
上記リグノセルロース系バイオマスは、微生物の培養または酵素による加水分解に供する前に、糖化効率を高めるための前処理を行うことが好ましい。前処理としては、例えば、脱水処理または乾燥処理、粉砕処理、および蒸煮処理などの従来公知の技術を適宜採用することができる。特に、稲わら、麦わら、とうもろこしの茎葉、コーンコブなどの農作物の非可食部などのリグノセルロース系バイオマスを用いる場合は、まず、風乾によりバイオマス中に含まれる水分を除く。その後、切断装置や粉砕装置を用いて、好ましくは１．０ｍｍ以下であり、より好ましくは０．０１〜１．０ｍｍであり、さらに好ましくは０．１〜１．０ｍｍの粉末状とすることが好ましい。 The lignocellulosic biomass is preferably subjected to a pretreatment for increasing the saccharification efficiency before being subjected to microorganism culture or enzymatic hydrolysis. As the pretreatment, for example, conventionally known techniques such as dehydration treatment or drying treatment, pulverization treatment, and steaming treatment can be appropriately employed. In particular, when using lignocellulosic biomass such as rice straw, wheat straw, corn stover, corn cob and other non-edible parts of agricultural products, the moisture contained in the biomass is first removed by air drying. Then, it is preferably 1.0 mm or less, more preferably 0.01 to 1.0 mm, and even more preferably 0.1 to 1.0 mm using a cutting device or a pulverizer. preferable.
上記リグノセルロース系バイオマスは、微生物の培養または酵素による加水分解に供するために、水性媒体に懸濁して用いる。水性媒体は微生物の増殖または酵素反応の進行が可能な範囲においては特に制限はないが、水、緩衝液、酸性水溶液、またはアルカリ性水溶液を用いることが好ましい。懸濁液中に含まれるリグノセルロース系バイオマスの粉砕物の濃度は、水性媒体に対して、通常、５０〜３００ｇ／Ｌであり、好ましくは１００〜２００ｇ／Ｌである。 The lignocellulosic biomass is used after being suspended in an aqueous medium in order to be subjected to microbial culture or enzymatic hydrolysis. The aqueous medium is not particularly limited as long as microbial growth or enzymatic reaction can proceed, but it is preferable to use water, a buffer solution, an acidic aqueous solution, or an alkaline aqueous solution. The density | concentration of the ground material of lignocellulosic biomass contained in suspension is 50-300 g / L normally with respect to an aqueous medium, Preferably it is 100-200 g / L.
上述の本発明の微生物を用いて糖化液を製造する場合、本発明の微生物は、上記懸濁液のみを培地とした場合であっても、十分な増殖能力を有するが、必要によっては炭素源、窒素源、または無機物などを懸濁液に補充したものを培地として用いることも可能である。これらの栄養源は、微生物の数を増やすための前培養の培地に添加してもよいし、本培養の培地に添加しても構わない。 When producing a saccharified solution using the above-described microorganism of the present invention, the microorganism of the present invention has sufficient growth ability even when only the above suspension is used as a medium, but if necessary, a carbon source. It is also possible to use a medium supplemented with a nitrogen source or an inorganic substance as a culture medium. These nutrient sources may be added to a preculture medium for increasing the number of microorganisms, or may be added to a main culture medium.
炭素源としては、例えば、グルコース、ガラクトース、フルクトース、およびキシロースなどの単糖、マルトース、ラクトース、およびスクロースなどの二糖、グリセリンおよびキシリトールなどの糖アルコールといった糖類が挙げられ、これらを単独であるいは２種以上を組み合わせて使用できる。特に、前培養の培地の培地にグルコースを添加することにより、本培養のときの微生物のリグノセルロース系バイオマスの分解活性をより向上させることができる。 Examples of the carbon source include saccharides such as monosaccharides such as glucose, galactose, fructose, and xylose, disaccharides such as maltose, lactose, and sucrose, and sugar alcohols such as glycerin and xylitol. Can be used in combination with more than one species. In particular, by adding glucose to the medium of the pre-culture medium, the degradation activity of the lignocellulosic biomass of the microorganism during the main culture can be further improved.
窒素源としては、例えば、アンモニア、塩化アンモニウム、硫酸アンモニウム、硝酸アンモニウムなどのアンモニウム塩、尿素、Ｌ−グルタミン酸などのアミノ酸類、あるいは尿酸などの無機あるいは有機の窒素化合物が使用できる。さらに、窒素源としては、ペプトン、ポリペプトン、肉エキス、酵母エキス、大豆加水分解物、大豆粉末、ミルクカゼイン、カザミノ酸、コーンスティープリカー（ＣＳＬ）などの窒素含有天然物を使用してもよい。これらのうち、塩化アンモニウム、硫酸アンモニウム、尿素、Ｌ−グルタミン酸などのアミノ酸類、尿酸などの無機あるいは有機窒素化合物、ペプトン、肉エキス、酵母エキスなの窒素含有天然物が好ましい。これらの窒素源は、単独であるいは２種以上を組み合わせて使用できる。これらの窒素源のうち、大豆粉末またはコーンスティープリカーを使用することが好ましい。 As the nitrogen source, for example, ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium nitrate, amino acids such as urea and L-glutamic acid, and inorganic or organic nitrogen compounds such as uric acid can be used. Furthermore, nitrogen-containing natural products such as peptone, polypeptone, meat extract, yeast extract, soybean hydrolysate, soybean powder, milk casein, casamino acid, corn steep liquor (CSL) may be used as the nitrogen source. Among these, amino acids such as ammonium chloride, ammonium sulfate, urea, and L-glutamic acid, and inorganic or organic nitrogen compounds such as uric acid, nitrogen-containing natural products such as peptone, meat extract, and yeast extract are preferable. These nitrogen sources can be used alone or in combination of two or more. Of these nitrogen sources, it is preferable to use soybean powder or corn steep liquor.
無機物としては、例えば、塩化ナトリウム、塩化カリウム、塩化カルシウム、リン酸カリウム、リン酸ナトリウム、硫酸マグネシウム、硫酸アンモニウムなどの、マグネシウム、マンガン、カルシウム、ナトリウム、カリウム、銅、鉄および亜鉛などのリン酸塩、塩酸塩、硫酸塩および酢酸塩などが用いられる。そのほか、チアミン、ビオチンなどのビタミン類、さらに必要に応じて、アデニン、ウラシルなどの核酸関連物質が使用されてもよい。これらの無機物は、単独であるいは２種以上を組み合わせて使用できる。 Examples of inorganic substances include sodium chloride, potassium chloride, calcium chloride, potassium phosphate, sodium phosphate, magnesium sulfate, ammonium sulfate, and other phosphates such as magnesium, manganese, calcium, sodium, potassium, copper, iron, and zinc. Hydrochloride, sulfate, acetate and the like are used. In addition, vitamins such as thiamine and biotin, and if necessary, nucleic acid-related substances such as adenine and uracil may be used. These inorganic substances can be used alone or in combination of two or more.
微生物の培養方法は、本発明の微生物の増殖が実質的に阻害されず、リグノセルロース系バイオマスから単糖類を生産可能な範囲であれば、特に制限なく当業者によって適宜調整されうるが、培養温度は、通常２０〜５０℃であり、好ましくは２８〜３７℃である。また、培地のｐＨは、通常３〜９であり、好ましくは４〜７である。培養前または培養中にｐＨが４未満となる場合は、アンモニアなどを用いてｐＨ制御を行うことが好ましい。本発明の微生物を培養する場合は、一定量の酸素を供給することが必要である。供給方法は、特に制限はないが、フラスコ培養の場合は回転振盪による攪拌、バイオリアクターを用いる培養の場合は通気攪拌をすることによって培地に酸素を供給する。 The method for culturing microorganisms can be appropriately adjusted by those skilled in the art without particular limitation as long as the growth of the microorganism of the present invention is not substantially inhibited and monosaccharides can be produced from lignocellulosic biomass. Is usually 20 to 50 ° C., preferably 28 to 37 ° C. Moreover, the pH of a culture medium is 3-9 normally, Preferably it is 4-7. When the pH is less than 4 before or during the culture, it is preferable to control the pH using ammonia or the like. When culturing the microorganism of the present invention, it is necessary to supply a certain amount of oxygen. The supply method is not particularly limited, but oxygen is supplied to the medium by stirring by rotary shaking in the case of flask culture and by aeration stirring in the case of culture using a bioreactor.
一方、本発明の酵素を用いた加水分解反応は、リグノセルロース系バイオマスを含む懸濁液中に酵素を添加し、懸濁液を攪拌下、インキュベートすることによって行われうる。懸濁液に添加する酵素量は、懸濁するリグノセルロース系バイオマスの粉砕物の質量に対して、好ましくは０．１〜３質量％、より好ましくは１〜２質量％である。かような量の酵素を用いることによって、通常１２〜１４４時間、好ましくは２４〜７２時間で加水分解反応が完了しうる。また、加水分解条件は、用いる酵素の至適条件を用いることが好ましい。ｐＨは通常３〜６であり、好ましくは３．５〜５である。また、反応温度は通常３０〜６０℃であり、好ましくは４０〜６０℃である。 On the other hand, the hydrolysis reaction using the enzyme of the present invention can be carried out by adding the enzyme to a suspension containing lignocellulosic biomass and incubating the suspension with stirring. The amount of enzyme added to the suspension is preferably 0.1 to 3% by mass, more preferably 1 to 2% by mass, based on the mass of the pulverized lignocellulosic biomass. By using such an amount of the enzyme, the hydrolysis reaction can be completed usually in 12 to 144 hours, preferably in 24 to 72 hours. Moreover, it is preferable to use the optimal conditions of the enzyme to be used for hydrolysis conditions. The pH is usually 3-6, preferably 3.5-5. Moreover, reaction temperature is 30-60 degreeC normally, Preferably it is 40-60 degreeC.
微生物の培養、または酵素による加水分解により得られた糖化液は、糖化液中の不溶物を適宜ろ過してもよいし、そのままの状態で、微生物代謝産物（エタノール、乳酸などの有用化合物）を産生する他の微生物の培養に供しても構わない。撹拌効率および収率向上の観点の観点からは、糖化液中の不溶物を予めろ過することが好ましい。 In the saccharified solution obtained by culturing microorganisms or hydrolyzing with enzymes, insolubles in the saccharified solution may be appropriately filtered, or microbial metabolites (useful compounds such as ethanol and lactic acid) may be used as they are. You may use for culture | cultivation of the other microorganisms to produce. From the viewpoint of improving stirring efficiency and yield, it is preferable to filter insolubles in the saccharified solution in advance.
他の微生物としては、特に制限はないが、例えば、乳酸を産生するＲｉｚｏｐｕｓ属、イタコン酸を生産するＡｓｐｅｒｇｉｌｌｕｓ ｔｅｒｒｅｕｓ、エタノールを産生するＰａｃｈｙｓｏｌｅｎ ｔａｎｎｏｐｈｉｌｕｓ、フマル酸を産生するＲｈｉｚｏｐｕｓ ｏｒｙｚａｅ、リンゴ酸を産生するＡｓｐｅｒｇｉｌｌｕｓ ｆｌａｖｕｓ、ピルビン酸を産生するＴｏｒｕｌｏｐｓｉｓ ｇｌａｂｒａｔａ、キシリトールを産生するＣａｎｄｉｄａ ｔｒｏｐｉｃａｌｉｓが挙げられる。本発明によって得られる糖化液を含む倍中でこれらの微生物を培養することによって、燃料や化成品を工業的規模で製造するが可能である。 Other microorganisms are not particularly limited, for example, Risopus genus that produces lactic acid, Aspergillus terreus that produces itaconic acid, Pachysolen tanophilus that produces ethanol, Rhizopus oryzae that produces fumaric acid, Aspergillus that produces malic acid flavus, Torulopsis grabrata producing pyruvate, and Candida tropicalis producing xylitol. By culturing these microorganisms in a fold containing the saccharified solution obtained by the present invention, it is possible to produce fuels and chemical products on an industrial scale.
本発明の作用効果を、以下の実施例および比較例を用いて説明する。ただし、本発明の技術的範囲が以下の実施例のみに制限されるわけではない。 The effect of this invention is demonstrated using a following example and a comparative example. However, the technical scope of the present invention is not limited only to the following examples.
＜菌株Ｍ１および菌株Ｍ２の稲わら分解活性の評価＞
［実施例１］
１．生産菌の培養
（前培養）
２００ｍｌ三角フラスコに下記表１５に示す組成のＣ培地２０ｍｌを入れ、オートクレーブで１２１℃、２０分間滅菌した。該Ｃ培地に菌株Ｍ１および菌株Ｍ２をそれぞれ１白金耳ずつ接種し、これを恒温振盪培養装置を用いて３２℃、回転数２２０ｐｐｍで２４時間振盪培養した（混合培養）。
<Evaluation of rice straw decomposition activity of strains M1 and M2>
[Example 1]
1. Production bacteria culture (pre-culture)
20 ml of C medium having the composition shown in Table 15 below was placed in a 200 ml Erlenmeyer flask and sterilized at 121 ° C. for 20 minutes in an autoclave. The C medium was inoculated with one platinum loop of each of strains M1 and M2, and this was cultured with shaking at 32 ° C. and a rotation speed of 220 ppm for 24 hours using a constant temperature shaking culture apparatus (mixed culture).
(本培養)
次に、下記表１６の組成を有する各培地を調製した。下記表１６において、対象なる成分が含まれている場合を「○」であらわし、含まれていない場合を「×」で表す。
(Main culture)
Next, each medium having the composition shown in Table 16 below was prepared. In Table 16 below, the case where the target component is included is represented by “◯”, and the case where it is not included is represented by “X”.
なお、上記表１６における稲わらは、粒径が１．０ｍｍ未満の粉砕物を用いた。稲わらの粉砕方法は以下のとおりである。稲わらを室温で約２ヵ月間風乾し、金切ばさみで１〜５ｃｍ程度に切断した。そして、これを家庭用ミキサーに５分間かけることにより粗粉砕した。該粗粉砕物をさらにボールミル（ＡＮＳ−６０Ｓ、日陶科学社製）を用いて微粉化し、稲わら粉砕物を得た。 In addition, the rice straw in the said Table 16 used the ground material whose particle size is less than 1.0 mm. The method for pulverizing rice straw is as follows. The rice straw was air-dried at room temperature for about 2 months and cut into about 1 to 5 cm with gold cutting shears. And this was coarsely pulverized by applying to a home mixer for 5 minutes. The coarsely pulverized product was further pulverized using a ball mill (ANS-60S, manufactured by Nichido Kagaku Co., Ltd.) to obtain a rice straw pulverized product.
次に、上記の培地１〜６を２００ｍｌ三角フラスコにそれぞれ２０ｍｌずつ入れ、オートクレーブで１２１℃、２０分間滅菌した。該培地１〜６に、上記前培養液を７（ｖ／ｖ）％植菌した。これを恒温振盪培養装置を用いて３２℃、回転数２２０ｐｐｍで６日間振盪培養した。 Next, 20 ml of each of the above media 1 to 6 was placed in a 200 ml Erlenmeyer flask and sterilized at 121 ° C. for 20 minutes in an autoclave. 7% (v / v)% of the preculture was inoculated into the media 1-6. This was shake-cultured for 6 days at 32 ° C. and a rotation speed of 220 ppm using a constant temperature shaking culture apparatus.
２．稲わら分解活性の評価（糖化）
上述の方法で得た粒径が１．０ｍｍ未満の稲わら粉砕物２ｇを水道水９ｍｌに懸濁した。該懸濁液に、上記培地１〜６で培養した培養液、またはそのろ液を希釈した酵素液１ｍｌを加え、５０℃で２４時間糖化した。この後、それぞれの糖化液をろ過し、得られたろ液中に含まれる全糖量をフェノール硫酸法により測定した。そして、稲わら分解活性を下記式（１）より算出した。なお、酵素活性は１［Ｕ］は、（１／６０）［μｋａｔ］に相当する。
2. Evaluation of rice straw decomposition activity (saccharification)
2 g of crushed rice straw having a particle size of less than 1.0 mm obtained by the above method was suspended in 9 ml of tap water. To the suspension, 1 ml of an enzyme solution obtained by culturing the above-described culture mediums 1 to 6 or a filtrate thereof was added and saccharified at 50 ° C. for 24 hours. Thereafter, each saccharified solution was filtered, and the total amount of sugar contained in the obtained filtrate was measured by the phenol-sulfuric acid method. And rice straw decomposition activity was computed from following formula (1). In the enzyme activity, 1 [U] corresponds to (1/60) [μkat].
［比較例１］
菌株Ｍ１および菌株Ｍ２に変えて、トリコデルマ・リーゼイ（Ｔｒｉｃｏｄｅｒｍａ ｒｅｅｓｅｉ）ＮＢＲＣ３１３２９を用いたことを除いては、実施例１と同様の方法で稲わら分解活性を求めた。
[Comparative Example 1]
Rice straw decomposition activity was determined in the same manner as in Example 1 except that Trichoderma reesei NBRC31329 was used instead of strains M1 and M2.
結果を下記表１７に示す。 The results are shown in Table 17 below.
表１７の結果より、本発明の菌株Ｍ１およびＭ２は、従来優れたセルラーゼ産生菌として知られるトリコデルマ・リーゼイ ＮＢＲＣ３１３２９よりも顕著な稲わら分解活性能を有することが示された。 From the results of Table 17, it was shown that the strains M1 and M2 of the present invention have a remarkable rice straw decomposition activity ability than Trichoderma reesei NBRC31329, which is conventionally known as an excellent cellulase-producing bacterium.
また、前培養の培地に、窒素源として大豆粉を、炭素源としてグルコースを添加することにより、本培養のときの微生物の増殖を促進し、微生物のリグノセルロース系バイオマスの分解活性をより向上させることができることが示された。 In addition, by adding soybean flour as a nitrogen source and glucose as a carbon source to the preculture medium, the growth of microorganisms during main culture is promoted and the decomposition activity of lignocellulosic biomass of microorganisms is further improved. It was shown that it can.
＜酵素活性の評価＞
［実施例２］
１．酵素の調製
（前培養）
２００ｍｌ三角フラスコに下記表１８に示す組成のＤ培地２０ｍｌを入れ、オートクレーブで１２１℃、２０分間滅菌した。該Ｄ培地に菌株Ｍ１および菌株Ｍ２をそれぞれ１白金耳ずつ接種し、これを恒温振盪培養装置を用いて３２℃、回転数２２０ｐｐｍで２４時間振盪培養した（混合培養）。
<Evaluation of enzyme activity>
[Example 2]
1. Enzyme preparation (pre-culture)
A 200 ml Erlenmeyer flask was charged with 20 ml of D medium having the composition shown in Table 18 below, and sterilized at 121 ° C. for 20 minutes in an autoclave. The D medium was inoculated with one platinum loop of each of the strains M1 and M2, and this was cultured with shaking at 32 ° C. and a rotation speed of 220 ppm for 24 hours using a constant temperature shaking culture apparatus (mixed culture).
（本培養）
次に、３Ｌジャーファーメンター（３ＭＤＬ型、丸菱バイオエンジ社製）に下記表１９に示す組成のＥ培地１．５Ｌを入れ、オートクレーブで１２１℃、２０分間滅菌した。該Ｂ培地に、上記で培養したＤ培地を７（ｖ／ｖ）％植菌した。これを、恒温振盪培養装置を用いて３２℃、回転数６００ｐｐｍ、通気量２ｖｖｍで６日間培養した。
(Main culture)
Next, 1.5 L of E medium having the composition shown in Table 19 below was placed in a 3 L jar fermenter (3MDL type, manufactured by Maruhishi Bioengineer), and sterilized by autoclaving at 121 ° C. for 20 minutes. The B medium was inoculated with 7 (v / v)% of the D medium cultured as described above. This was cultured for 6 days at 32 ° C., a rotation speed of 600 ppm, and an aeration rate of 2 vvm using a constant temperature shaking culture apparatus.
上記の６日間の培養中、培養開始から０（培養開始直後）、２４、４８、７２、および８４時間後の培養液を１ｍｌ取り出し、上記実施例１の３．稲わら分解活性の評価と同様の方法で、各時間の培養液の稲わら分解活性を評価した。結果を下記表２０に示す。 During the above 6-day culture, 1 ml of the culture solution at 0 (immediately after the start of culture), 24, 48, 72, and 84 hours after the start of the culture was taken out, and the above-mentioned 3. The rice straw decomposing activity of the culture solution at each time was evaluated in the same manner as the evaluation of rice straw decomposing activity. The results are shown in Table 20 below.
６日間培養後の培養液をろ過し、ケーキ（残渣）を水洗した。ろ液とケーキの水洗液とをあわせて濃縮乾固した。次に、この濃縮乾固物を５℃の冷水２００ｍｌに溶解し、不溶分をろ別した後、これにアセトンを１０００ｍｌ加えて、アセトン沈殿物を得た。そして、これを凍結乾燥して酵素（「酵素Ｍ１Ｍ２」と称する）を得た。なお、上記の各工程で得られた生成物の収量、酵素活性または比活性、総活性、および収率を下記表２１に示す。なお、酵素活性は上記実施例１の分解活性の評価と同様の方法を用いて測定した。 The culture solution after 6 days of culture was filtered, and the cake (residue) was washed with water. The filtrate and the cake washings were combined and concentrated to dryness. Next, this concentrated dry solid was dissolved in 200 ml of cold water at 5 ° C., insoluble matter was filtered off, 1000 ml of acetone was added thereto, and an acetone precipitate was obtained. This was freeze-dried to obtain an enzyme (referred to as “enzyme M 1 M 2 ”). The yield, enzyme activity or specific activity, total activity, and yield of the product obtained in each of the above steps are shown in Table 21 below. The enzyme activity was measured using the same method as the evaluation of the degradation activity in Example 1 above.
２．酵素化学的性質の評価
（安定性に対する温度の影響）
０．０５Ｍ酢酸バッファー（ｐＨ５．５）９ｍｌに、上記で得た酵素Ｍ１Ｍ２１ｇを溶解したものを酵素溶液とした。ふた付試験管に該酵素溶液１ｍｌと、上記酢酸バッファー９ｍｌとを入れ、それぞれ所定の温度で１０時間振盪した後、試験管を５℃に冷却した。ついで、この試験管に上記実施例１で調製した稲わら粉末（粒径１．０ｍｍ未満のもの）を１ｇ加え、５０℃で２４時間糖化反応を行った。この後、それぞれの糖化液をろ過し、得られたろ液中に含まれる全糖量をフェノール硫酸法により測定した。そして、分解活性（糖化活性）を上記式（１）より算出した。得られた値のうち最も高い安定性を与えた１０℃における分解活性に対する、それぞれの温度における活性を百分率で表した。結果を図１に示す。
2. Enzymatic chemistry evaluation (temperature effect on stability)
An enzyme solution was prepared by dissolving 1 g of the enzyme M 1 M 2 obtained above in 9 ml of 0.05 M acetate buffer (pH 5.5). 1 ml of the enzyme solution and 9 ml of the acetic acid buffer were placed in a test tube with a lid, shaken at a predetermined temperature for 10 hours, and then the test tube was cooled to 5 ° C. Next, 1 g of rice straw powder (particle size less than 1.0 mm) prepared in Example 1 was added to the test tube, and saccharification reaction was performed at 50 ° C. for 24 hours. Thereafter, each saccharified solution was filtered, and the total amount of sugar contained in the obtained filtrate was measured by the phenol-sulfuric acid method. And decomposition | disassembly activity (saccharification activity) was computed from the said Formula (1). The activity at each temperature was expressed as a percentage of the decomposition activity at 10 ° C. that gave the highest stability among the obtained values. The results are shown in FIG.
（糖化活性に対する温度の影響）
ふた付試験管に、上記酵素溶液１ｍｌと、上記酢酸バッファー９ｍｌとを入れ、上記実施例１で調製した稲わら粉末（粒径１．０ｍｍ未満のもの）を１ｇ加え、所定の温度で２４時間糖化反応を行った。この後、それぞれの糖化液をろ過し、得られたろ液中に含まれる全糖量をフェノール硫酸法により測定した。そして、分解活性（糖化活性）を上記式（１）より算出した。得られた値のうち最も高い安定性を与えた５５℃における分解活性に対する、それぞれの温度における活性を百分率で表した。結果を図１に示す。
(Effect of temperature on saccharification activity)
In a test tube with a lid, 1 ml of the enzyme solution and 9 ml of the acetate buffer are added, 1 g of rice straw powder (particle size less than 1.0 mm) prepared in Example 1 is added, and the mixture is heated at a predetermined temperature for 24 hours. A saccharification reaction was performed. Thereafter, each saccharified solution was filtered, and the total amount of sugar contained in the obtained filtrate was measured by the phenol-sulfuric acid method. And decomposition | disassembly activity (saccharification activity) was computed from the said Formula (1). The activity at each temperature was expressed as a percentage of the decomposition activity at 55 ° C. that gave the highest stability among the obtained values. The results are shown in FIG.
（安定性に対するｐＨの影響）
ふた付試験管に上記酵素溶液１ｍｌと、それぞれ所定のｐＨに調整した０．０５Ｍ酢酸バッファー９ｍｌを入れ、５０℃で１０時間振盪した後、試験管を５℃に冷却した。ついで、この試験管に上記実施例１で調製した稲わら粉末（粒径１．０ｍｍ未満のもの）を１ｇ加え、５０℃で２４時間糖化反応を行った。この後、それぞれの糖化液をろ過し、得られたろ液中に含まれる全糖量をフェノール硫酸法により測定した。そして、分解活性（糖化活性）を上記式（１）より算出した。得られた値のうち最も高い安定性を与えたｐＨ３．７における分解活性に対する、それぞれのｐＨにおける活性を百分率で表した。結果を図２に示す。
(Effect of pH on stability)
In a test tube with a lid, 1 ml of the enzyme solution and 9 ml of 0.05M acetic acid buffer each adjusted to a predetermined pH were placed, shaken at 50 ° C. for 10 hours, and then cooled to 5 ° C. Next, 1 g of rice straw powder (particle size less than 1.0 mm) prepared in Example 1 was added to the test tube, and saccharification reaction was performed at 50 ° C. for 24 hours. Thereafter, each saccharified solution was filtered, and the total amount of sugar contained in the obtained filtrate was measured by the phenol-sulfuric acid method. And decomposition | disassembly activity (saccharification activity) was computed from the said Formula (1). The activity at each pH was expressed as a percentage of the degradation activity at pH 3.7 that gave the highest stability among the obtained values. The results are shown in FIG.
（糖化活性に対する温度の影響）
ふた付試験管に、上記酵素溶液１ｍｌと、それぞれ所定のｐＨに調整した０．０５Ｍ酢酸バッファー９ｍｌを入れ、上記実施例１で調製した稲わら粉末（粒径１．０ｍｍ未満のもの）を１ｇ加え、５０℃で２４時間糖化反応を行った。この後、それぞれの糖化液をろ過し、得られたろ液中に含まれる全糖量をフェノール硫酸法により測定した。そして、分解活性（糖化活性）を上記式（１）より算出した。得られた値のうち最も高い安定性を与えたｐＨ４．５における分解活性に対する、それぞれのｐＨにおける活性を百分率で表した。結果を図２に示す。
(Effect of temperature on saccharification activity)
In a test tube with a lid, 1 ml of the enzyme solution and 9 ml of 0.05 M acetic acid buffer adjusted to a predetermined pH were added, and 1 g of rice straw powder (particle size less than 1.0 mm) prepared in Example 1 was used. In addition, a saccharification reaction was performed at 50 ° C. for 24 hours. Thereafter, each saccharified solution was filtered, and the total amount of sugar contained in the obtained filtrate was measured by the phenol-sulfuric acid method. And decomposition | disassembly activity (saccharification activity) was computed from the said Formula (1). The activity at each pH was expressed as a percentage of the degradation activity at pH 4.5 that gave the highest stability among the obtained values. The results are shown in FIG.
［実施例３］
Ｄ培地に接種した微生物を菌株Ｍ１のみとしたことを除いては、実施例２と同様の方法で酵素を調製した。得られた酵素を「酵素Ｍ１」と称する。そして、実施例２と同様の方法で酵素Ｍ１の比活性を測定した。
[Example 3]
An enzyme was prepared in the same manner as in Example 2 except that the strain M1 was the only microorganism inoculated into the D medium. The obtained enzyme is referred to as “enzyme M 1 ”. Then, to measure the specific activity of the enzyme M 1 in the same manner as in Example 2.
［実施例４］
Ｄ培地に接種した微生物を菌株Ｍ２のみとしたことを除いては、実施例２と同様の方法で酵素を調製した。得られた酵素を「酵素Ｍ２」と称する。そして、実施例２と同様の方法で酵素Ｍ２の比活性を測定した。
[Example 4]
The enzyme was prepared in the same manner as in Example 2 except that the strain M2 was the only microorganism inoculated into the D medium. The obtained enzyme is referred to as “enzyme M 2 ”. Then, to measure the specific activity of the enzyme M 2 in the same manner as in Example 2.
［比較例２］
アクレモニウムセルラーゼ（明治製菓社製）を用いて、実施例２と同様の方法で比活性を測定した。
[Comparative Example 2]
Specific activity was measured in the same manner as in Example 2 using Acremonium cellulase (Meiji Seika Co., Ltd.).
［比較例３］
メイセラーゼ（明治製菓社製）を用いて、実施例２と同様の方法で比活性を測定した。
[Comparative Example 3]
Specific activity was measured in the same manner as in Example 2 using Meicerase (manufactured by Meiji Seika Co., Ltd.).
［比較例４］
スミチーム（新日本化学工業社製）を用いて、実施例２と同様の方法で比活性を測定した。
[Comparative Example 4]
Specific activity was measured in the same manner as in Example 2 using Sumiteam (manufactured by Shin Nippon Chemical Industry Co., Ltd.).
酵素の比活性の結果を下記表２２に示す。 The results of the specific activity of the enzyme are shown in Table 22 below.
表２２の結果より、本発明の酵素Ｍ１Ｍ２、酵素Ｍ１、および酵素Ｍ２は、従来のセルラーゼよりも優れた稲わら分解活性を有することが示された。 From the results of Table 22, it was shown that the enzyme M 1 M 2 , the enzyme M 1 , and the enzyme M 2 of the present invention have rice straw decomposition activity superior to that of conventional cellulases.
＜酵素Ｍ１Ｍ２を用いた稲わら糖化液の製造、および該糖化液を用いたＬ−乳酸の製造＞
［実施例５］
３Ｌジャーファーメンター（ＭＤＬ−３型、丸菱バイオエンジ社製）に水道水１．０Ｌを入れ、上記実施例１で調製した稲わら粉末（粒径１．０ｍｍ未満のもの）２００ｇを懸濁し、さらに水道水を加えて液量を１．５Ｌとした。該懸濁液のｐＨを、２５％塩酸を用いて４．５に調整した。そして、実施例２で得た酵素Ｍ１Ｍ２を２０Ｕ／ｍｌとなるように添加し、５０℃で攪拌（２００ｒｐｍ）下、７２時間糖化反応を行った。反応液中の全糖量、グルコース量、キシロース量、および糖化率の経時変化を図３に示す。
<Production of rice straw saccharified solution using enzyme M 1 M 2 and production of L-lactic acid using the saccharified solution>
[Example 5]
Into a 3L jar fermenter (MDL-3 type, manufactured by Maruhishi Bio-Engineering Co., Ltd.) is added 1.0 L of tap water, and 200 g of rice straw powder (particle size less than 1.0 mm) prepared in Example 1 is suspended. Further, tap water was added to make the liquid volume 1.5L. The pH of the suspension was adjusted to 4.5 using 25% hydrochloric acid. Then, the enzyme M 1 M 2 obtained in Example 2 was added to a 20 U / ml, stirred at 50 ° C. (200 rpm) under at 72 hours saccharification reaction. FIG. 3 shows changes over time in the total sugar amount, glucose amount, xylose amount, and saccharification rate in the reaction solution.
得られた糖化液を３Ｌジャーファーメンターに仕込み、これにリン酸一カリウムを０．３（ｇ／Ｌ）、硫酸マグネシウムを０．２５（ｇ／Ｌ）となるように添加した。該糖化液をオートクレーブを用いて１２１℃で１０分間滅菌した。次に、予め３００ｍｌ三角フラスコで前培養したリゾプス エスピー（Ｒｉｚｏｐｕｓ ｓｐ．）ＭＫ９６−１１５６（受託番号：ＦＥＲＭ ＢＰ−６７７７）の培養液１００ｍｌを添加し、回転数３００ｒｐｍ、通器量０．５ｖｖｍ、培養温度３０℃で７２時間培養した。培養中、２５％アンモニア水を用いて培養液のｐＨを６．０に制御した。全糖量、グルコース量、およびＬ−乳酸量の経時変化を図４に示す。リゾプス エスピー ＭＫ９６−１１５６（受託番号：ＦＥＲＭ ＢＰ−６７７７）の培養により、全糖９０ｇ／Ｌから４２ｇ／ＬのＬ−乳酸が得られた。また、稲わら粉末からのＬ−乳酸の収率は２１（ｗ／ｗ）％であった。 The resulting saccharified solution was charged into a 3 L jar fermenter, and monopotassium phosphate was added to 0.3 (g / L) and magnesium sulfate was added to 0.25 (g / L). The saccharified solution was sterilized at 121 ° C. for 10 minutes using an autoclave. Next, 100 ml of a culture solution of Risopus sp. MK96-1156 (accession number: FERM BP-6777) pre-cultured in a 300 ml Erlenmeyer flask was added, the rotation speed was 300 rpm, the flow rate was 0.5 vvm, the culture temperature. The cells were cultured at 30 ° C. for 72 hours. During the culture, the pH of the culture solution was controlled at 6.0 using 25% aqueous ammonia. FIG. 4 shows changes with time in the total sugar amount, glucose amount, and L-lactic acid amount. By culturing Rhizopus SP MK96-1156 (Accession Number: FERM BP-6777), L-lactic acid of 90 g / L to 42 g / L of total sugar was obtained. The yield of L-lactic acid from rice straw powder was 21 (w / w)%.
＜酵素Ｍ１Ｍ２を用いたコーンコブ糖化液の製造、および該糖化液を用いたＬ−乳酸の製造＞
［実施例６］
稲わら粉末に変えて、上述の［１次スクリーニング］で調製したコーンコブ粉末（粒径１．０ｍｍ未満のもの）を用い、糖化時間を４８時間、Ｒｉｚｏｐｕｓ ｓｐ． ＭＫ９６−１１５６（受託番号：ＦＥＲＭ ＢＰ−６７７７）の培養時間を９６時間としたことを除いては、実施例５と同様の方法で、Ｌ−乳酸を生産した。糖化反応液中の全糖量、グルコース量の経時変化を図５に示す。また、Ｒｉｚｏｐｕｓ ｓｐ． ＭＫ９６−１１５６（受託番号：ＦＥＲＭ ＢＰ−６７７７）の培養液中の全糖量、グルコース量、およびＬ−乳酸量の経時変化を図６に示す。Ｒｉｚｏｐｕｓ ｓｐ． ＭＫ９６−１１５６（受託番号：ＦＥＲＭ ＢＰ−６７７７）の培養により、全糖１０２ｇ／Ｌから４６ｇ／ＬのＬ−乳酸が得られた。また、コーンコブ粉末からのＬ−乳酸の収率は２１（ｗ／ｗ）％であった。
<Manufacture of corn cob saccharified solution using enzyme M 1 M 2 and manufacture of L-lactic acid using the saccharified solution>
[Example 6]
Instead of the rice straw powder, the corn cob powder (particle size less than 1.0 mm) prepared in the above-mentioned [Primary screening] was used, the saccharification time was 48 hours, and Rizopus sp. L-lactic acid was produced in the same manner as in Example 5 except that the culture time of MK96-1156 (accession number: FERM BP-6777) was 96 hours. FIG. 5 shows changes over time in the total sugar amount and glucose amount in the saccharification reaction solution. In addition, Risopus sp. FIG. 6 shows changes over time in the total sugar amount, glucose amount, and L-lactic acid amount in the culture solution of MK96-1156 (Accession Number: FERM BP-6777). Risopus sp. By culturing MK96-1156 (accession number: FERM BP-6777), L-lactic acid having a total sugar of 102 g / L to 46 g / L was obtained. Moreover, the yield of L-lactic acid from corn cob powder was 21 (w / w)%.
＜稲わら糖化液を用いたエタノールの製造＞
硫酸玉つきガラス管を備えた１００ｍｌ三角フラスコに、実施例５と同様の方法で製造した稲わら糖化液および下記表２３に示す添加物を加えた培養液２０ｍｌを準備した。これにパン酵母（オリエンタルドライイースト、オリエンタル酵母工業社製）０．５ｇを加え、３０℃で静置培養を行った。経時的にフラスコ全体の重量を測定し、その重量の減少量を下記式（２）にあてはめ、各培養時間後のエタノール量を算出した。下記式（２）において、フラスコの重量の減少量はエタノール発酵による炭酸ガス発生量（ｇ）を意味する。アルコール発酵により、グルコース１モルからエタノール２モルと炭酸ガス（ＣＯ２
）２モルが生産されることから、炭酸ガス発生量（ｇ）に１．０４７（４６．０６８／４４．００９５）を乗じて得られる値が、エタノール量（ｇ）となる。そして、培養開始前の糖化液中のグルコース量に対するエタノール量を百分率で表し、各培養時間におけるエタノール収率（％）とした。結果を下記表２３に示す。
<Production of ethanol using rice straw saccharified solution>
A 100 ml Erlenmeyer flask equipped with a glass tube with a sulfate ball was prepared 20 ml of a culture solution prepared by adding the rice straw saccharified solution produced in the same manner as in Example 5 and the additives shown in Table 23 below. To this, 0.5 g of baker's yeast (Oriental Dry Yeast, manufactured by Oriental Yeast Co., Ltd.) was added, and static culture was performed at 30 ° C. The weight of the entire flask was measured over time, the amount of decrease in the weight was applied to the following formula (2), and the amount of ethanol after each culture time was calculated. In the following formula (2), the amount of decrease in the weight of the flask means the amount of carbon dioxide generated (g) by ethanol fermentation. By alcohol fermentation, 1 mol of glucose to 2 mol of ethanol and carbon dioxide (CO 2
) Since 2 moles are produced, a value obtained by multiplying the carbon dioxide generation amount (g) by 1.047 (46.068 / 44.0095) is the amount of ethanol (g). And the amount of ethanol with respect to the amount of glucose in the saccharified solution before the start of culture was expressed as a percentage, and was defined as the ethanol yield (%) in each culture time. The results are shown in Table 23 below.
表２３の結果より、本発明の酵素Ｍ１Ｍ２を用いて製造された糖化液は、酵母エキス、乾燥酵母、尿素、硫安などの窒素源を補填することのなく、４６％の高収率でエタノールを製造できることが示された。 From the results in Table 23, the saccharified solution produced using the enzyme M 1 M 2 of the present invention has a high yield of 46% without supplementing nitrogen sources such as yeast extract, dry yeast, urea, ammonium sulfate, etc. It was shown that ethanol can be produced.
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