JP2009033982A - Efficient weight reduction of mowed lawn grass by enzymic saccharification - Google Patents
Efficient weight reduction of mowed lawn grass by enzymic saccharification Download PDFInfo
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Abstract
Description
本発明は、シバを刈り込んだ後に廃棄物として生じる刈屑を減量することを目的に、刈芝を酵素によって糖化しエネルギー資源として有効活用可能な形態にする刈芝の減量方法に関するものである。また、得られた糖化液をエタノールや乳酸などに変換する方法に関するものである。 The present invention relates to a method for reducing the amount of cut grass that is saccharified by an enzyme and made into a form that can be effectively used as an energy resource, for the purpose of reducing the amount of cutting waste generated as waste after cutting the shiba. The present invention also relates to a method for converting the obtained saccharified solution into ethanol, lactic acid or the like.
環境省資料「市区町村別のゴルフ場面積」（地域ＰＲＴＲ非点源排出量推定マニュアル第一版）によると、ゴルフ場は全国で１３．２万ｈａ余りに広がり、日本国土（３７２８万ｈａ）の０．３６％、農業可能地域面積（１７２７万ｈａ）の０．７７％を占める（環境省環境統計集）。ゴルフ場で植栽されたシバは、保全のため定期的に刈り込み整備することが必要であり、その結果シバの刈屑が生じる。これらの刈芝の総量は統計的に調査されていないが、農産廃棄物（農作物非食用部）が年約１３００万トン（バイオマスニッポン、主なバイオマスの発生量と利用状況の変化）であることを考えると、年間で１０万トン程度の刈屑が産業廃棄物として排出されていると推算される。これらの刈屑は個々のゴルフ場で収集され、そのままあるいは天日乾燥させた後、可燃性廃棄物として焼却処分されているが、この処分に要する費用と労力は莫大である。また、刈屑を焼却することにより生じる二酸化炭素は温室効果ガスとして地球温暖化の原因となっているとも言われており、焼却処分量の減量は産業的にも地球環境的にも早期に解決が求められる課題である。これらの課題を解決することを目的とした、刈芝の処分方法の１つとして、例えば、セルラーゼを利用した刈芝分解促進剤が公知である（特許文献１）。 According to the Ministry of the Environment document “Golf Area by Municipality” (Regional PRTR Non-point Source Emission Estimate Manual 1st Edition), the golf course has spread over 130,000 ha nationwide, Japan (37.28 million ha) Occupy 0.76% of the agricultural area (17.27 million ha) (Ministry of the Environment's Environmental Statistics Collection). The buckwheat planted at the golf course needs to be pruned and maintained regularly for conservation, resulting in shiba cutting waste. The total amount of these turf grasses has not been statistically investigated, but agricultural waste (agricultural crop non-edible part) is about 13 million tons a year (biomass Japan, changes in the amount of biomass generated and usage) Therefore, it is estimated that about 100,000 tons of cutting waste are discharged as industrial waste annually. These swarf are collected at individual golf courses, and are incinerated as flammable waste as it is or after being dried in the sun, but the cost and labor required for this disposal are enormous. In addition, carbon dioxide generated by incineration of cutting waste is said to cause global warming as a greenhouse gas, and the reduction of incineration disposal amount can be resolved at an early stage both industrially and globally. Is a required issue. As one of the methods for disposing of mowing lawn for the purpose of solving these problems, for example, a lawn lawn decomposition accelerator using cellulase is known (Patent Document 1).
一方、近年、石油代替可能な資源として再生可能なバイオマスを原料としたバイオリファイナリー技術が着目されている中で、イネ科植物であるシバは草本系バイオマスとして注目すべき重要な原材料の一つと考えることができる。 On the other hand, in recent years, with the focus on biorefinery technology that uses renewable biomass as an alternative to petroleum, grasses are considered to be one of the important raw materials to be noted as herbaceous biomass. be able to.
バイオマスの主成分はグルコースが直鎖状に連なったセルロースであるが、一般にバイオマス中のセルロースはヘミセルロースやリグニンなどの副成分に囲まれており単離が困難である。従ってこれらの資材を利用する場合には、バイオマスそのものをランダムに分解し必要な糖を選択的に取得・利用する方法、あるいはバイオマスから選択的にセルロースのみを効率よく分解する方法を利用する方法が採用されている。 The main component of biomass is cellulose in which glucose is linearly linked, but generally cellulose in biomass is surrounded by subcomponents such as hemicellulose and lignin, and is difficult to isolate. Therefore, when using these materials, there is a method of randomly decomposing biomass itself and selectively acquiring and using the necessary sugar, or a method of efficiently decomposing only cellulose from biomass. It has been adopted.
前者の方法としては、これまでにバイオマスそのものを濃硫酸あるいは希硫酸にて加水分解し糖を得る方法が知られているが、この場合、リグニン等の分解あるいは基質の酸化による副反応が生じて分解液中に副生成物が蓄積し、得られた糖の利用が阻害される場合がある。また大量の酸を使用することは、反応廃液の処分において環境負担が大きく、新たな産業廃棄物として水質保全法や工場排水規制法、資源有効利用促進法等の規則に準じた適切な処分が必要となり、産業廃棄物の低減に繋がらないといった問題がある。一方、後者の方法としては、セルロースのみを選択的に分解できる酵素セルラーゼを用いて加水分解する方法があり、酸加水分解法に比べ穏和な条件でグルコールを得ることができると共に、不要な副産物の生成を極力抑制できることから、以降の工程での糖利用効率が向上することが期待できる。 As the former method, there is known a method in which biomass itself is hydrolyzed with concentrated sulfuric acid or dilute sulfuric acid to obtain a sugar, but in this case, a side reaction occurs due to decomposition of lignin or oxidation of the substrate. By-products may accumulate in the decomposition solution and use of the resulting sugar may be hindered. In addition, the use of a large amount of acid has a large environmental burden in the disposal of reaction waste liquid, and as a new industrial waste, appropriate disposal in accordance with regulations such as the Water Quality Conservation Law, the Industrial Wastewater Regulation Law, and the Law for Promotion of Effective Utilization of Resources is required. There is a problem that it is necessary and does not lead to reduction of industrial waste. On the other hand, as the latter method, there is a method in which hydrolysis is performed using an enzyme cellulase capable of selectively degrading only cellulose. Glucol can be obtained under mild conditions as compared with the acid hydrolysis method, and unnecessary by-products can be obtained. Since the production can be suppressed as much as possible, it can be expected that the sugar utilization efficiency in the subsequent steps is improved.
しかし、前述の通り、バイオマス中のセルロースはヘミセルロースやリグニンなどの副成分に囲まれているため、そのままセルラーゼを作用させても効率の良い糖化は困難である。従って、バイオマスには適切な前処理を施す必要がある。 However, as described above, since cellulose in biomass is surrounded by subcomponents such as hemicellulose and lignin, efficient saccharification is difficult even if cellulase is allowed to act as it is. Therefore, the biomass needs to be pretreated appropriately.
酵素糖化の前処理法としては、アルカリや希酸などの化学薬品を使用する処理法が知られているが、前述の酸加水分解にて述べた通り、装置の腐食の問題および反応廃液の処分が大きな問題である。また薬品を用いない方法として、物理的に処理する方法が幾つか知られている。例えば、高温・高圧での蒸煮・爆砕法は、高圧下でバイオマスに高温水を噴霧した後急激に減圧することでバイオマスの組織構造を破壊する方法であり、多くの報告例があるが、ヘミセルロースの回収が悪く、将来的にヘミセルロースも有効に活用する場合に不利となる可能性が示されている。また電子線処理は実験室スケールでの例証しかなく、実用化の可能性は未だ低い状況にある。 As a pretreatment method for enzymatic saccharification, a treatment method using a chemical such as alkali or dilute acid is known, but as mentioned in the above-mentioned acid hydrolysis, the problem of corrosion of the apparatus and disposal of the reaction waste liquid Is a big problem. In addition, as a method that does not use chemicals, several methods of physical treatment are known. For example, the steaming / explosion method at high temperature and high pressure is a method of destroying the structure of biomass by spraying high temperature water on the biomass under high pressure and then rapidly reducing the pressure. It has been shown that there is a possibility that it may be disadvantageous when the hemicellulose is also effectively used in the future. Moreover, electron beam processing is only an example on a laboratory scale, and the possibility of practical use is still low.
本発明の課題は、ゴルフ場などで大量に発生する刈芝の減量方法を提供することにある。また、本発明は、ゴルフ場などで大量に発生する刈芝の減量を目的に、産業廃棄物として焼却処分するのではなく、有用なバイオマスとして利用するために、酵素による効率的な糖化技術を確立し有効利用することを目的とする。 An object of the present invention is to provide a method for reducing the amount of cut grass that is generated in large quantities at a golf course or the like. In addition, the present invention provides an efficient enzyme saccharification technology for use as useful biomass rather than incineration as industrial waste for the purpose of reducing the amount of grass that is generated in large quantities at golf courses and the like. The purpose is to establish and use effectively.
前記課題は、本発明による、刈り取ったシバを乾燥させた後粉砕し、次いで粉砕済みのシバをセルラーゼにより糖化することを特徴とする、刈芝の減量方法により解決することができる。また、本発明の好ましい態様によれば、刈り取ったシバから得られた糖化液を微生物処理することによりエタノール発酵あるいは乳酸発酵を行う。 The above-mentioned problem can be solved by the method for reducing the amount of mowed grass according to the present invention, characterized in that the cut barn is dried and then pulverized, and then the crushed barn is saccharified with cellulase. Moreover, according to the preferable aspect of this invention, ethanol fermentation or lactic acid fermentation is performed by carrying out the microorganism process of the saccharified liquid obtained from the harvested buckwheat.
本発明者は、上記の課題を解決すべく鋭意研究を重ねた結果、乾燥させた刈芝を有用なセルロース系バイオマスとして使用するために、適切なサイズに粉砕する工程と、粉砕した刈芝を適切な条件にてセルラーゼにて糖化する工程により、硫酸や基質酸化物等の不純物を含まない糖を効率的に得ることに成功した。
この結果、産業廃棄物として焼却されてきた刈芝を減量すると共に、エタノールや乳酸等の有用物質へ変換し活用することが可能となる。
As a result of intensive studies to solve the above problems, the present inventor has pulverized the cut grass to a suitable size in order to use the dried cut grass as a useful cellulosic biomass. Through the process of saccharification with cellulase under appropriate conditions, we succeeded in efficiently obtaining sugars free from impurities such as sulfuric acid and substrate oxides.
As a result, it is possible to reduce the amount of cut grass that has been incinerated as industrial waste and to convert it into useful substances such as ethanol and lactic acid.
本発明の減量方法は、乾燥工程、粉砕工程、及び糖化工程を少なくとも含む。
本発明の乾燥工程は、次工程の粉砕工程において刈芝を粉砕可能となる程度に乾燥可能であれば、その手段及び乾燥の程度は限定されるものではないが、粉砕工程における粒度の管理又は雑菌繁殖の抑制の観点から、適切に乾燥させることが好ましい。乾燥方法は天日乾燥でもよく、またヒーターや温風等を用いた機器（乾燥機）でもよい。エネルギー消費を避けるために、天日乾燥が好ましい。乾燥の程度は、刈芝の含水量として好ましくは２５％以下、更に好ましくは１０％以下が適切である。
The weight loss method of the present invention includes at least a drying step, a pulverizing step, and a saccharification step.
In the drying process of the present invention, the means and the degree of drying are not limited as long as mowing lawn can be pulverized in the subsequent pulverization process, but the particle size management in the pulverization process or It is preferable to dry appropriately from a viewpoint of suppression of miscellaneous bacteria propagation. The drying method may be sun drying or may be a device (dryer) using a heater, hot air, or the like. In order to avoid energy consumption, sun drying is preferred. The degree of drying is preferably 25% or less, more preferably 10% or less as the moisture content of the cut grass.
粉砕工程では、乾燥させた刈芝を、引き続き粉末状に粉砕する。大量の刈芝を粉砕処理するため使用する粉砕機としては、例えば、スクリーン型、ファン型、衝撃型、ローターミル型、ボールミル型、ピンミル型、カッティングミル型、気流式、剪断式など多様な粉砕機の使用が可能である。粉砕品の粒度は、例えば、粒径１０００μｍ（１６メッシュ）以下であり、２５０μｍ（６０メッシュ）以下であることが好ましい。粒度の下限は、所望の粒径範囲に応じて選択可能な粉砕機の性能により適宜決定されるため、特に限定されるものではないが、例えば、粒径１μｍ以上であり、１０μｍ以上であることが好ましい。 In the pulverization step, the dried cut grass is continuously pulverized into powder. Various crushers used for crushing a large amount of mowing grass include, for example, screen type, fan type, impact type, rotor mill type, ball mill type, pin mill type, cutting mill type, airflow type, shearing type, etc. The machine can be used. The particle size of the pulverized product is, for example, a particle size of 1000 μm (16 mesh) or less, and preferably 250 μm (60 mesh) or less. The lower limit of the particle size is appropriately determined depending on the performance of the pulverizer that can be selected according to the desired particle size range, and is not particularly limited. For example, the particle size is 1 μm or more and 10 μm or more. Is preferred.
本発明では、前記粉砕処理により、薬品等による前処理を行わなくても刈芝を効率的に糖化することが可能となるため、環境負荷を低減することが可能となる。 In the present invention, the pulverization process enables efficient saccharification of cut grass without pretreatment with chemicals or the like, thereby reducing the environmental load.
糖化工程で使用するセルラーゼ、すなわち、粉砕処理した刈芝の糖化に使用する酵素は、セルロースを分解してグルコースを遊離することのできる酵素である限り、特に限定されるものではないが、セルロース繊維をランダムに分解する活性（例えば、アビセラーゼ活性）、若しくは、セルロース繊維を末端から分解する活性（例えば、β−グルコシダーゼ活性）の少なくとも一方（好ましくは両方）の活性を有するセルラーゼを用いることができ、前記活性の少なくとも一方（好ましくは両方）の活性が高い酵素がより好ましい。 The cellulase used in the saccharification step, that is, the enzyme used for saccharification of the pulverized turf lawn is not particularly limited as long as it is an enzyme capable of decomposing cellulose and releasing glucose. A cellulase having at least one activity (preferably both) of an activity that randomly degrades (for example, avicellase activity) or an activity that degrades cellulose fibers from the end (for example, β-glucosidase activity), An enzyme having a high activity of at least one (preferably both) of the activities is more preferred.
また、粉砕された刈芝のセルロース利用効率を更に向上するためには、セルロース繊維を囲むリグニンやヘミセルロース等を効率的に除去することは有効である。従って、本発明の糖化工程においては、前記セルラーゼが、セルロース繊維を囲む副成分（例えば、リグニンやヘミセルロース等）を分解する活性を更に有することが好ましい。 In order to further improve the cellulose utilization efficiency of the pulverized cut grass, it is effective to efficiently remove lignin, hemicellulose and the like surrounding the cellulose fibers. Therefore, in the saccharification step of the present invention, it is preferable that the cellulase further has an activity of decomposing subcomponents (for example, lignin and hemicellulose) surrounding the cellulose fiber.
使用するセルラーゼとしては、これらの条件に該当していれば、通常市販されているセルラーゼ製剤を使用することが可能である。また、単一のセルラーゼ製剤ではこれらの条件に該当しなくても、複数のセルラーゼ製剤を適切に混合することにより条件に該当するのであれば、使用することが可能である。
セルロース繊維をランダムに分解する活性（例えば、アビセラーゼ活性）、セルロース繊維を末端から分解する活性（例えば、β−グルコシダーゼ活性）、リグニン及びヘミセルロース等の分解活性のいずれも高いセルラーゼ製剤としては、例えば、アクレモニウムセルラーゼ（明治製菓株式会社製）を用いることができる。また、同様の性能を持つセルラーゼ製剤であれば、その他の市販のセルラーゼ製剤を使用することも可能である。市販のセルラーゼ製剤としては、具体的には、セルラーゼＡ、セルラーゼＴ、ヘミセルラーゼアマノ９０（天野エンザイム社製）、ＧＯＤＯ−ＴＣＬ、ＧＯＤＯ−ＴＣＤ−Ｈ（合同酒精社製）、セルライザー、セルラーゼＸＬ−５２２（ナガセケムテックス社製）、セルクラスト（ノボザイムズ社製）、セルロシンＡＣ４０、セルロシンＴ２、セルロシンＴＰ２５（ヒチビィアイ社製）、セルラーゼオノズカ３Ｓ（ヤクルト薬品工業社製）、スミチームＡＣ、スミチームＣ、スミチームＸ（新日本化学工業社製）、メイセラーゼ（明治製菓社製）等を挙げることができる。更に、基質である刈芝粉砕品の状態に応じ、酵素活性を補強するために、アビセラーゼ活性やβ−グルコシダーゼ活性に優れたセルラーゼを適宜添加することで、効率の良い糖化を得ることができる。
As the cellulase to be used, a commercially available cellulase preparation can be used as long as these conditions are met. Moreover, even if it does not correspond to these conditions in a single cellulase preparation, it can be used as long as the conditions are satisfied by appropriately mixing a plurality of cellulase preparations.
Cellulase preparations having high activity for randomly degrading cellulose fibers (for example, avicellase activity), activity for degrading cellulose fibers from the end (for example, β-glucosidase activity), lignin, hemicellulose, etc. Acremonium cellulase (Meiji Seika Co., Ltd.) can be used. In addition, other commercially available cellulase preparations can be used as long as they have the same performance. Specific examples of commercially available cellulase preparations include cellulase A, cellulase T, hemicellulase Amano 90 (manufactured by Amano Enzyme), GODO-TCL, GODO-TCD-H (manufactured by Godo Shusei Co., Ltd.), cell riser, and cellulase XL. -522 (manufactured by Nagase ChemteX Corp.), cell crust (manufactured by Novozymes), cellulosin AC40, cellulosin T2, cellulosin TP25 (manufactured by Hichibiai), cellulase Onozuka 3S (manufactured by Yakult Pharmaceutical Co., Ltd.), Sumiteam AC, Sumiteam C, Sumiteam X (manufactured by Shin Nippon Chemical Industry Co., Ltd.), Meicelase (manufactured by Meiji Seika Co., Ltd.) and the like can be mentioned. Furthermore, efficient saccharification can be obtained by appropriately adding cellulase excellent in avicelase activity and β-glucosidase activity in order to reinforce the enzyme activity according to the state of the pulverized lawn turf as a substrate.
糖化工程において刈芝粉砕品の効率的糖化を行うために、セルラーゼ製剤の高活性の維持、活性安定化のためには、糖化反応液中のｐＨを酵素の至適条件に調整することが好ましい。このためには、例えば、５〜１００ｍｍｏｌ／ＬのｐＨ３．５〜５．５に調製した緩衝液、例えば、クエン酸緩衝液あるいは酢酸緩衝液を使用することができる。あるいは、望ましくは緩衝液を使用せず、反応槽にｐＨを調整する設備を据え付けた上で水を使用することが適切である。また、糖化反応の反応温度は、使用酵素に応じて適宜決定することができ、例えば、温度を３０〜６０℃に調整した上で、好ましくは撹拌しながら、糖化反応を実施することができる。 In order to efficiently saccharify the pulverized lawn turf in the saccharification step, it is preferable to adjust the pH in the saccharification reaction solution to the optimum enzyme conditions in order to maintain the high activity of the cellulase preparation and stabilize the activity . For this purpose, for example, a buffer solution adjusted to pH 3.5 to 5.5 of 5 to 100 mmol / L, for example, a citrate buffer solution or an acetate buffer solution can be used. Alternatively, it is preferable to use water after installing a device for adjusting pH in the reaction tank, desirably without using a buffer solution. Moreover, the reaction temperature of saccharification reaction can be suitably determined according to the enzyme used, for example, after adjusting temperature to 30-60 degreeC, Preferably saccharification reaction can be implemented, stirring.
糖化反応の効率化において重要な因子である基質と酵素の比率は、一定濃度の刈芝粉砕品に対しセルラーゼ濃度を低下すると、酵素当たりの還元糖量生成量は見かけ上向上するが、刈芝粉砕品あたりの還元糖量生成量は低下するため、刈芝の減量に繋がらない。刈芝粉砕品に一定比率以上で糖化可能な適切な酵素量を添加することが望ましい。 The ratio of substrate to enzyme, which is an important factor in improving the efficiency of the saccharification reaction, is that the amount of reducing sugar produced per enzyme is apparently improved when the cellulase concentration is reduced compared to the pulverized turf at a certain concentration. Since the amount of reducing sugar produced per pulverized product decreases, it does not lead to a reduction in cut grass. It is desirable to add an appropriate amount of enzyme that can be saccharified to a chopped turf product at a certain ratio or higher.
糖化反応を実施した後の酵素反応液は、糖化の結果生じた可溶性の糖類（グルコース、ヘミセルロース由来のキシロース、アラビノース、マンノース等の糖類等）および不溶性の刈芝残渣が含まれるため、所望により、不溶物の分別を行うことができる。分別の方法は、固形分と液体分が効率的に分離できる方法であれば特に限定されるものではなく、例えば、フィルタープレスやメンブランフィルター（ＭＦ膜）、セラ膜を用いた濾過や遠心分離器での分別により実施することができる。処理する反応液の液量によりフィルタープレスのような濾過機やメンブランフィルター（ＭＦ膜）を使用する場合と、遠心分離機を使用する場合があり、適宜最適な機器の選定を実施することが望ましい。 The enzyme reaction solution after the saccharification reaction contains soluble saccharides (glucose, saccharides such as glucose, hemicellulose-derived xylose, arabinose, mannose, etc.) and insoluble turf residue as a result of saccharification. Insoluble matter can be separated. The separation method is not particularly limited as long as the solid content and the liquid content can be efficiently separated. For example, filtration using a filter press, a membrane filter (MF membrane), a cera membrane, or a centrifuge It can be carried out by fractionation. Depending on the amount of the reaction solution to be treated, a filter such as a filter press or a membrane filter (MF membrane) may be used, or a centrifuge may be used. .
回収した糖液は用途に応じて適切な濃度に濃縮し、使用することができる。例えば、そのまま酵母によるアルコール発酵や乳酸菌による乳酸発酵に使用する場合には、固形分含量として１０〜２５％Ｗ／Ｖに濃縮することができ、保管等する場合には５０〜７０％まで濃縮、あるいは更に濃縮し結晶化することができる。微生物としては、エタノール発酵の場合には、例えば、酵母を用いることができ、乳酸発酵の場合には、例えば、乳酸菌を用いることができる。 The recovered sugar solution can be concentrated to an appropriate concentration according to the application and used. For example, when it is used as it is for alcoholic fermentation by yeast or lactic acid fermentation by lactic acid bacteria, it can be concentrated to 10 to 25% W / V as a solid content, and concentrated to 50 to 70% when stored, Alternatively, it can be further concentrated and crystallized. In the case of ethanol fermentation, for example, yeast can be used as the microorganism, and in the case of lactic acid fermentation, for example, lactic acid bacteria can be used.
一方、除去した刈芝残渣は、例えば、天日等により乾燥することができる。乾燥した刈芝残渣は、例えば、産業廃棄物として処分することができるが、上記糖の回収により、固形分重量は当初の４０〜７０％に減量できるため、産業廃棄物の減量につなぐことができる。 On the other hand, the removed mowing lawn residue can be dried by, for example, sunlight. The dried turf residue can be disposed of as industrial waste, for example, but the solid content can be reduced to 40-70% of the original weight by recovering the sugar, leading to the reduction of industrial waste. it can.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。 EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.
《実施例１：アクレモニウムセルラーゼ（ＡＣＥ）による刈芝の糖化》
基質である刈芝は、家庭用ミルミキサーによって粉砕処理後、６０メッシュの篩を用いて粒度２５０μｍ以下に調整した。ｐＨ４．５に調整した２０ｍｍｏｌ／Ｌクエン酸緩衝液４５ｍＬに対し、粉砕処理した刈芝を１ｇとなるように添加・懸濁し、糖化の基質とした。同緩衝液５ｍＬに、カルボキシメチルセルラーゼ（ＣＭＣａｓｅ）活性で２５０Ｕ相当のアクレモニウムセルラーゼ（ＡＣＥ；明治製菓株式会社製）を溶解した上で、基質懸濁液に添加し、基質の沈降を防止する目的で反応中は往復振盪機を用いて毎分１５０回転で振盪し、反応温度５０℃にて反応を継続した。反応開始後、４、８、１２、３６、７２、及び９６時間目に反応液の一部を抜き取り、糖化反応によって生成した還元糖及びグルコース濃度の測定を行った。
<< Example 1: Saccharification of cut grass with acremonium cellulase (ACE) >>
The cut grass as a substrate was pulverized by a home mill mixer and then adjusted to a particle size of 250 μm or less using a 60 mesh sieve. To 45 mL of 20 mmol / L citrate buffer adjusted to pH 4.5, pulverized cut grass was added and suspended to 1 g, and used as a substrate for saccharification. The purpose of preventing the sedimentation of the substrate by dissolving acrylmethyl cellulase (ACE; manufactured by Meiji Seika Co., Ltd.) equivalent to 250 U in carboxymethyl cellulase (CMCase) activity in 5 mL of the same buffer and adding it to the substrate suspension During the reaction, the mixture was shaken at 150 rpm using a reciprocating shaker, and the reaction was continued at a reaction temperature of 50 ° C. At 4, 8, 12, 36, 72, and 96 hours after the start of the reaction, a part of the reaction solution was withdrawn, and the reducing sugar and glucose concentrations produced by the saccharification reaction were measured.
結果を表１並びに図１及び図２に示す。ＡＣＥの糖化作用によって刈芝生成した還元糖量は、反応後４時間目に０．２５ｇに達し、更に、９６時間目には０．３４５ｇに到達した。また、その中に含まれるＤ−グルコース量は、それぞれ０．１５ｇ及び０．２０ｇに達した。 The results are shown in Table 1 and FIGS. The amount of reducing sugar produced by the ACE saccharification action reached 0.25 g at 4 hours after the reaction, and further reached 0.345 g at 96 hours. Moreover, the amount of D-glucose contained therein reached 0.15 g and 0.20 g, respectively.
酸加水分解法によって、刈芝中の主な糖質として、セルロース３９．８％、Ｄ−キシロース１６．７％、その他の糖質９．６％が含まれていることが判明している。これより、刈芝を完全に糖化することによって生成すると期待される全還元糖量及びグルコース量は次の式によって算出される。
全グルコース量＝セルロース含有量×１８０／（１８０−１８）
全還元糖量＝全グルコース含有量＋キシロース含有量＋その他の糖質含量
The acid hydrolysis method has been found to contain 39.8% cellulose, 16.7% D-xylose, and 9.6% other carbohydrates as the main carbohydrates in mowing lawn. From this, the total reducing sugar amount and glucose amount expected to be generated by completely saccharifying the cut grass are calculated by the following equations.
Total glucose amount = cellulose content × 180 / (180-18)
Total reducing sugar content = total glucose content + xylose content + other carbohydrate content
すなわち、刈芝１ｇ中の全還元糖量は０．７０５ｇ、全グルコース量は０．４４２ｇであると算出されることから、ＡＣＥでの糖化によって得られた還元糖及びグルコースは、それぞれ刈芝中の４９．６％及び４４．１％の収率であると確認することができた。また、この糖化反応により、刈芝１ｇは残渣０．６５ｇに減量できることが示された。 That is, since the total reducing sugar amount in 1 g of cut grass is calculated to be 0.705 g and the total glucose amount is 0.442 g, reducing sugar and glucose obtained by saccharification with ACE are respectively in the cutting grass. It was confirmed that the yields were 49.6% and 44.1%. Moreover, it was shown that 1 g of cut grass can be reduced to 0.65 g of residue by this saccharification reaction.
《実施例２：フミコーラ・インソレンス由来のエンドグルカナーゼ（ＥＧ）による刈芝の糖化》
使用する糖化酵素としてフミコーラ・インソレンス（Humicola insolens）由来のエンドグルカナーゼ（ＥＧ，明治製菓株式会社製）を用い、同様の糖化実験を実施した。結果を表２並びに図１及び図２に示すが、アクレモニウムセルラーゼとは明らかに異なる糖化反応の推移を示したものの、最終的には同レベルの糖化を得ることができた。このことから、糖化酵素の種類によらず、最終的に添加基質の３３．２％が糖質として可溶化され、残渣の減量につながることが確認できた。
<< Example 2: Saccharification of cut grass with endoglucanase (EG) derived from Humicola insolens >>
Similar saccharification experiments were carried out using endoglucanase (EG, manufactured by Meiji Seika Co., Ltd.) derived from Humicola insolens as the saccharifying enzyme to be used. The results are shown in Table 2 and FIG. 1 and FIG. 2, and although the transition of the saccharification reaction was clearly different from that of Acremonium cellulase, the same level of saccharification was finally obtained. From this, it was confirmed that 33.2% of the added substrate was finally solubilized as a saccharide regardless of the type of saccharifying enzyme, leading to a reduction in the amount of residue.
《実施例３：ＡＣＥ及びＥＧの併用による刈芝糖化の効率化》
刈芝の糖化効率の更なる向上のために、上記２種類の性質の異なるセルラーゼの併用を行った。実施例１と同様に調製した基質懸濁液に、ＡＣＥとＥＧを等量混合しＣＭＣアーゼ活性で２５０Ｕとなるよう調整・添加し、９６時間糖化反応を行った。
結果（９６時間反応）を表２に示す。アクレモニウムセルラーゼ又はＥＧの単独使用に対し約１．５倍の０．４６９ｇの還元糖を得ることができた。また、Ｄ−グルコースの遊離量も０．２５８ｇと単独使用の１．５〜１．７倍の生成量であった。更に酵素の使用量を増量したところ、１０００Ｕの添加で還元糖生成量が０．５８９ｇまで向上した。
<< Example 3: Efficiency of cut grass saccharification by combined use of ACE and EG >>
In order to further improve the saccharification efficiency of cut grass, cellulases having two different properties were used in combination. ACE and EG were mixed in an equal amount to the substrate suspension prepared in the same manner as in Example 1, adjusted and added so that the CMCase activity was 250 U, and saccharified for 96 hours.
The results (96 hours reaction) are shown in Table 2. It was possible to obtain 0.469 g of reducing sugar, which is about 1.5 times as much as Acremonium cellulase or EG alone. In addition, the amount of D-glucose released was 0.258 g, which was 1.5 to 1.7 times the amount used alone. When the amount of the enzyme used was further increased, the amount of reducing sugar produced increased to 0.589 g by adding 1000 U.
また、２５０Ｕ添加条件において、基質の添加量を増量したところ、表３（７２時間反応）に示すように、添加量増に従い糖化によって得られる還元糖量も増量することが示された。一方、得られた還元糖量を加えた基質１ｇ当たりで換算すると、５ｇ添加区が最も高い糖化効率を示すことが確認できた。 In addition, when the amount of substrate added was increased under the 250U addition condition, it was shown that the amount of reducing sugar obtained by saccharification increased as the amount added increased as shown in Table 3 (72 hour reaction). On the other hand, when converted per 1 g of the substrate to which the amount of reducing sugar obtained was added, it was confirmed that the 5 g addition group showed the highest saccharification efficiency.
《実施例４：粒度の違いによる粉砕刈芝の効率糖化》
刈芝の糖化効率の更なる向上のために、刈芝を粉砕するための粒度の影響を調査した。刈芝はスクリーン式粉砕機を用い、９０μｍ以下、９０−１２５μｍ、１２５−２５０μｍ、２５０−５００μｍに調整し、実施例１と同様に基質懸濁液を調製した。酵素液は、実験例３の結果良好な糖化を示した、ＡＣＥとＥＧを等量混合しＣＭＣアーゼ活性で２５０Ｕとなるよう調整した酵素を用い、２８時間糖化反応を行った。
結果（２８時間反応）を表４に示す。糖化による還元糖およびグルコースの生成量は、刈芝の粒度が２５０μｍ以下であれば大きな差は認められないが、２５０−５００μｍまで粒度が大きくなると、糖の生成量に大きく影響を受けることが示された。
<< Example 4: Efficient saccharification of pulverized cut grass by difference in particle size >>
In order to further improve the saccharification efficiency of the cut grass, the effect of the particle size for grinding the cut grass was investigated. Karishiba was adjusted to 90 μm or less, 90-125 μm, 125-250 μm, 250-500 μm using a screen grinder, and a substrate suspension was prepared in the same manner as in Example 1. As an enzyme solution, an enzyme prepared by mixing ACE and EG in equal amounts and adjusted to have a CMCase activity of 250 U, which showed good saccharification as a result of Experimental Example 3, was subjected to saccharification reaction for 28 hours.
The results (28 hour reaction) are shown in Table 4. The amount of reducing sugar and glucose produced by saccharification is not significantly different if the particle size of mowing lawn is 250 μm or less, but it is shown that when the particle size is increased to 250-500 μm, the amount of sugar produced is greatly affected. It was done.
本発明の減量方法は、産業廃棄物処理の用途に適用することができる。 The weight loss method of the present invention can be applied to industrial waste treatment.
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