JPH04341183A - Chemically modified cellulase - Google Patents
Chemically modified cellulaseInfo
- Publication number
- JPH04341183A JPH04341183A JP3111959A JP11195991A JPH04341183A JP H04341183 A JPH04341183 A JP H04341183A JP 3111959 A JP3111959 A JP 3111959A JP 11195991 A JP11195991 A JP 11195991A JP H04341183 A JPH04341183 A JP H04341183A
- Authority
- JP
- Japan
- Prior art keywords
- cellulase
- copolymer
- activity
- modification
- modified
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010059892 Cellulase Proteins 0.000 title claims abstract description 45
- 229940106157 cellulase Drugs 0.000 title claims abstract description 45
- 229920001577 copolymer Polymers 0.000 claims abstract description 14
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 6
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims abstract description 5
- ATVJXMYDOSMEPO-UHFFFAOYSA-N 3-prop-2-enoxyprop-1-ene Chemical compound C=CCOCC=C ATVJXMYDOSMEPO-UHFFFAOYSA-N 0.000 claims description 5
- 125000000217 alkyl group Chemical group 0.000 claims description 4
- 125000005702 oxyalkylene group Chemical group 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 abstract description 34
- 108090000790 Enzymes Proteins 0.000 abstract description 34
- 229940088598 enzyme Drugs 0.000 abstract description 33
- 230000000694 effects Effects 0.000 abstract description 30
- 238000006243 chemical reaction Methods 0.000 abstract description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 abstract description 17
- 238000012986 modification Methods 0.000 abstract description 17
- 230000004048 modification Effects 0.000 abstract description 17
- 239000007864 aqueous solution Substances 0.000 abstract description 5
- 125000000539 amino acid group Chemical group 0.000 abstract description 3
- 125000003277 amino group Chemical group 0.000 abstract description 3
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical group O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 abstract description 3
- 238000003756 stirring Methods 0.000 abstract description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 abstract 2
- 238000000034 method Methods 0.000 description 13
- 108010084185 Cellulases Proteins 0.000 description 10
- 102000005575 Cellulases Human genes 0.000 description 10
- 229920000642 polymer Polymers 0.000 description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- 230000007423 decrease Effects 0.000 description 8
- 239000003960 organic solvent Substances 0.000 description 7
- 238000011282 treatment Methods 0.000 description 7
- 229920002678 cellulose Polymers 0.000 description 6
- 239000001913 cellulose Substances 0.000 description 6
- 238000000354 decomposition reaction Methods 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 230000000977 initiatory effect Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 241000233866 Fungi Species 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 238000007334 copolymerization reaction Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 108090000317 Chymotrypsin Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- GOOHAUXETOMSMM-UHFFFAOYSA-N Propylene oxide Chemical compound CC1CO1 GOOHAUXETOMSMM-UHFFFAOYSA-N 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 102000035124 heme enzymes Human genes 0.000 description 1
- 108091005655 heme enzymes Proteins 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
Classifications
-
- 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
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
Abstract
Description
【０００１】0001
【産業上の利用分野】この発明は修飾酵素に係り、より
詳細には、セルロ−ス分解酵素であるセルラ−ゼをポリ
マ−で化学修飾した化学修飾セルラ−ゼに関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to modified enzymes, and more particularly to chemically modified cellulases obtained by chemically modifying cellulases, which are cellulose-degrading enzymes, with polymers.
【０００２】0002
【従来の技術】酵素は、それが示す反応特異性により、
生体触媒の一つとして種々の反応に利用されている。し
かしながら、酵素を単独で使用するこということは、酵
素活性の持続安定性、反応環境への適応性、さらに反応
終了後に酵素を回収して再び使用する再利用性などの実
用的見地からは、必ずしも有効ではない。[Prior Art] Enzymes, due to their reaction specificity,
It is used as a biocatalyst in various reactions. However, the use of an enzyme alone is difficult from a practical standpoint, such as long-term stability of enzyme activity, adaptability to the reaction environment, and reusability of collecting the enzyme and using it again after the completion of the reaction. Not necessarily valid.
【０００３】そこで、酵素を固定化して使用する技術が
種々考案されている。そのような例としては、共有結合
、物理的吸着、イオン結合、生化学的特異結合などを利
用して担体に結合させる担体結合法、格子状物質やマイ
クロカプセルで酵素を包み込む包括法、酵素同士を架橋
剤で結合させる架橋法などを挙げることができる。また
、これらの方法を組み合わせた複合法もある。さらに、
酵素を両親媒性合成高分子で修飾することにより有機溶
媒可溶化酵素とする方法も提案されており、ポリエチレ
ングリコ−ル（ＰＥＧ）で修飾したカタラ−ゼ、ペルオ
キシダ−ゼ、キモトリプシン、リパ−ゼ、ヘム酵素など
が報告されている。[0003] Various techniques have therefore been devised to immobilize and use enzymes. Examples of such methods include carrier binding methods that utilize covalent bonds, physical adsorption, ionic bonds, biochemical specific bonds, etc., entrapment methods that envelop enzymes in lattice-like materials or microcapsules, and methods that bind enzymes to carriers. Examples include a crosslinking method in which these are bonded together using a crosslinking agent. There is also a composite method that combines these methods. moreover,
A method has also been proposed to make enzymes solubilized in organic solvents by modifying them with amphiphilic synthetic polymers, such as catalase, peroxidase, chymotrypsin, and lipase modified with polyethylene glycol (PEG). , heme enzymes, etc. have been reported.
【０００４】しかしながら、植物の主要部分を形成する
セルロ−スを分解するセルラ−ゼについては、固定化や
化学修飾の研究事例がない。セルロ−スを分解して低分
子化することは、未利用有機資源の有効利用に直接結び
付く重要な技術である。例えば、セルロ−スの低分子化
はアルコ−ルへの変換の中間過程として有用であり、ま
た低分子化された物質は新たな原料として利用可能であ
る。[0004] However, there is no research on immobilization or chemical modification of cellulase, which decomposes cellulose, which forms the main part of plants. The decomposition of cellulose into low-molecular-weight molecules is an important technology that directly leads to the effective use of unused organic resources. For example, reducing the molecular weight of cellulose is useful as an intermediate process in the conversion to alcohol, and the reduced molecular weight substance can be used as a new raw material.
【０００５】セルロ−スを分解して低分子化するための
処理としては、従来、物理的処理としての粉砕を経た後
、強アルカリ処理、強酸処理等の化学的処理が行なわれ
ている。セルラ−ゼを用いた酵素処理も近年盛んに研究
され、多くの報告があるが、その多くはセルラ−ゼで総
称される酵素を精製分離して各成分の活性を調べる酵素
学的研究や、動植物、菌体等から新たなセルラ−ゼを得
る方向で進められている。また、高活性のセルラ−ゼを
産出するために、遺伝子操作も試みられてはいる。[0005] Conventionally, as a treatment for decomposing cellulose to reduce its molecular weight, a physical treatment such as pulverization is followed by a chemical treatment such as a strong alkali treatment or a strong acid treatment. Enzymatic treatments using cellulases have been actively researched in recent years, and there have been many reports, but most of them involve enzymatic research that purifies and separates enzymes collectively known as cellulases and examines the activity of each component, Progress is being made in the direction of obtaining new cellulases from animals, plants, fungi, etc. Genetic manipulation has also been attempted to produce highly active cellulases.
【０００６】[0006]
【発明が解決しようとする課題】セルロ−スの分解に酵
素を使用することは、分解後の処理を考えた場合、従来
の物理的および化学的処理による方法に比較して有利で
ある。しかしながら、酵素による分解は、反応速度が遅
いこと、酵素活性安定性が悪いこと、酵素が高価であり
、しかも再利用が難しいことなどが問題となっている。
特に、分解過程において、セルラ−ゼが基質であるセル
ロ−スに吸着してしまい活性を失ってしまうことが大き
な問題点であった。Problems to be Solved by the Invention The use of enzymes for the decomposition of cellulose is advantageous compared to conventional physical and chemical treatment methods when considering post-decomposition treatments. However, problems with enzymatic decomposition include slow reaction rate, poor enzyme activity stability, expensive enzymes, and difficulty in reusing. In particular, a major problem was that during the decomposition process, cellulase adsorbed to the substrate cellulose and lost its activity.
【０００７】これに関しては、界面活性剤とセルラ−ゼ
との併用による活性向上を目的とした、Ｃａｓｔａｎｏ
ｎら（Ｂｉｏｔｅｃｈｎｏｌ． Ｂｉｏｅｎｇ．， ２
３， １３６５ （１９８１））、Ｏｏｓｈｉｍａ ら
（ｉｂｉｄ．， ２８， １７２７ （１９８６））、
Ｐａｒｋ、Ｋａｊｉｕｃｈｉら（ｉｂｉｄ．，印刷中）
の研究があり、界面活性剤との併用は酵素活性の維持に
効果があるとされている。[0007] Regarding this, Castano's
(Biotechnol. Bioeng., 2
3, 1365 (1981)), Ooshima et al. (ibid., 28, 1727 (1986)),
Park, Kajiuchi et al. (ibid., in press)
Research has shown that the combination of surfactants is effective in maintaining enzyme activity.
【０００８】しかしながら、再利用という点からは、セ
ルラ−ゼが分離しやすい形態になっていることが望まし
い。また、固定化は、基質が固体もしくは高分子である
ため、適当な方法とはいえない。However, from the point of view of reuse, it is desirable that cellulase be in a form that can be easily separated. Furthermore, immobilization is not an appropriate method because the substrate is solid or polymeric.
【０００９】この発明は、活性安定性、ｐＨ安定性、耐
アルコ−ル性等の実用的見地から要求される諸性質が付
与され、かつ分離・回収を行ない易い形態であって再利
用が容易である化学修飾セルラ−ゼを提供することを目
的とする。[0009] This invention provides various properties required from a practical standpoint such as activity stability, pH stability, and alcohol resistance, and is in a form that is easy to separate and recover, making it easy to reuse. The purpose of the present invention is to provide a chemically modified cellulase.
【００１０】0010
【課題を解決するための手段】本発明者は、上記事情に
鑑み、鋭意研究の結果、ポリオキシアルキレングリコ−
ルアルキル（メタ）アリルエ−テル・無水マレイン酸共
重合体でセルラ−ゼを化学修飾することにより、前記目
的が達成されることを見出した。[Means for Solving the Problems] In view of the above circumstances, the present inventors, as a result of intensive research, have developed a polyoxyalkylene glycol.
It has been found that the above object can be achieved by chemically modifying cellulase with a ralkyl (meth)allyl ether/maleic anhydride copolymer.
【００１１】この発明の化学修飾セルラ−ゼにおいては
、ポリオキシアルキレングリコ−ルアルキル（メタ）ア
リルエ−テル・無水マレイン酸共重合体を修飾用高分子
として用いている。このような共重合体としては、例え
ば、下記化２に示される一般式（Ｉ）で表わされる共重
合体を挙げることができる。In the chemically modified cellulase of the present invention, a polyoxyalkylene glycol alkyl (meth)allyl ether/maleic anhydride copolymer is used as a modifying polymer. Examples of such a copolymer include a copolymer represented by the general formula (I) shown in Chemical Formula 2 below.
【００１２】0012
【化２】[Case 2]
【００１３】上記一般式（Ｉ）において、ＡＯはオキシ
アルキレン基、Ｒはアルキル基、ｎは重合度、およびｋ
は共重合度をそれぞれ表わす。上記一般式（Ｉ）におい
て、ＡＯで表わされるオキシアルキレン基としては、エ
チレンオキシド［−（ＣＨ２ ＣＨ２ Ｏ）− ］、プ
ロピレンオキシド［−（ＣＨ２ （ＣＨ３ ）ＣＨＯ）
− ］等を挙げることができる。
また、Ｒで表わされるアルキル基としては、炭素数 １
ないし１８のアルキル基が好ましい。さらに、重合度ｎ
は ５〜５０程度、および共重合度ｋは ５〜５０程度
がそれぞれ望ましい。In the above general formula (I), AO is an oxyalkylene group, R is an alkyl group, n is the degree of polymerization, and k
each represents the degree of copolymerization. In the above general formula (I), the oxyalkylene group represented by AO includes ethylene oxide [-(CH2 CH2 O)-], propylene oxide [-(CH2 (CH3)CHO)
- ] etc. In addition, the alkyl group represented by R has 1 carbon number
to 18 alkyl groups are preferred. Furthermore, the degree of polymerization n
is preferably about 5 to 50, and the degree of copolymerization k is preferably about 5 to 50.
【００１４】上記一般式（Ｉ）で表わされる共重合体に
ついては、特開昭６４−１０８号公報、特開昭６４−１
０９号公報、特開平１−２８７４１１号公報、特開平２
−１３８３１７号公報、特開平２−１３８３１８号公報
、特開平２−１３８３１９号公報、特開平２−１３８３
２０号公報、および特開昭６３−２２６３５８ 号公報
に詳細に記載されている。Regarding the copolymer represented by the above general formula (I), JP-A-64-108, JP-A-64-1
No. 09, JP-A-1-287411, JP-A-2
-138317, JP 2-138318, JP 2-138319, JP 2-1383
It is described in detail in Japanese Patent Publication No. 20 and Japanese Unexamined Patent Publication No. 63-226358.
【００１５】この発明において、化学修飾される酵素と
しては、一般にセルラ−ゼと総称されるセルロ−ス分解
酵素であればどのようなものでもよい。代表的なセルラ
−ゼとしては、Ｔｒｉｃｈｏｄｅｒｍａ 属や Ａｃｒ
ｅｍｏｎｉｕｍ 類等の糸状菌が産生するセルラ−ゼを
挙げることができる。[0015] In the present invention, the enzyme to be chemically modified may be any cellulose-degrading enzyme generally referred to as cellulase. Typical cellulases include Trichoderma genus and Acr.
Examples include cellulases produced by filamentous fungi such as C. monium.
【００１６】セルラ−ゼの修飾は、セルラ−ゼに存在す
るアミノ酸残基中のアミノ基（ −ＮＨ２ ）と修飾用
高分子中に存在する無水マレイン酸基との反応を利用す
ることにより行なうことができる。この修飾反応は、例
えば、温度 ５℃以下の条件で、適当濃度の酵素（粗酵
素でも精製酵素でもよい）水溶液と修飾用高分子とを緩
やかな撹拌条件下で徐々に混合して反応させることによ
り行なう。この際、反応ｐＨ は ８以上に保つことが
肝要である。修飾用高分子の最終使用量は、所望する修
飾度によって異なるが、通常、酵素量の ０〜 １００
倍である。修飾酵素は、反応終了後の状態でそのまま使
用することも可能であるが、必要に応じて、脱塩・凍結
乾燥処理後、粉末として保存することができ、さらに有
機溶媒を用いて精製して精製修飾酵素として保存するこ
ともできる。[0016] Modification of cellulase is carried out by utilizing the reaction between amino groups (-NH2) in amino acid residues present in cellulase and maleic anhydride groups present in the modifying polymer. Can be done. This modification reaction can be carried out by, for example, gradually mixing and reacting an aqueous solution of an enzyme (crude enzyme or purified enzyme) at an appropriate concentration with a modifying polymer under gentle stirring conditions at a temperature of 5°C or lower. This is done by At this time, it is important to maintain the reaction pH at 8 or higher. The final amount of the modifying polymer used varies depending on the desired degree of modification, but is usually 0 to 100% of the amount of enzyme.
It's double. The modified enzyme can be used as it is after the reaction is completed, but if necessary, it can be stored as a powder after desalting and freeze-drying, and it can be further purified using an organic solvent. It can also be stored as a purified modified enzyme.
【００１７】[0017]
実施例１
修飾用高分子としてＡＫＭ−０５３１ およびＡＫＭ−
１５１１ を用いて、市販のセルラ−ゼ（ヤクルト社、
オノズカＲ−１０ ）を ０℃の条件下で修飾した。Ａ
ＫＭ−０５３１およびＡＫＭ−１５１１ は日本油脂（
株）から市販されているポリオキシアルキレングリコ−
ルアルキル（メタ）アリルエ−テル・無水マレイン酸共
重合体であり、いずれも上記一般式（Ｉ）で表わされる
構造を有している。上記一般式（Ｉ）における、それぞ
れのＲ、ＡＯ、ｎおよびｋは以下の通りである。Example 1 AKM-0531 and AKM- as modification polymers
1511, commercially available cellulase (Yakult Co., Ltd.,
Onozuka R-10) was modified under 0°C conditions. A
KM-0531 and AKM-1511 are manufactured by NOF (
Polyoxyalkylene glyco, commercially available from
It is a ralkyl (meth)allyl ether/maleic anhydride copolymer, and both have a structure represented by the above general formula (I). In the above general formula (I), each R, AO, n and k are as follows.
【００１８】
ＡＫＭ−０５３
１ ＡＫＭ−１５１１
Ｒ ＣＨ３
ＣＨ３ ＡＯ
ＣＨ２ ＣＨ２ Ｏ ＣＨ２ ＣＨ２
Ｏ ｎ
９ ３０
ｋ ３０
１０
ＭＷ １８，０００
１６，０００ それぞれの高分子を用いて
得られた修飾セルラ−ゼの特性を調べた。その結果を以
下に示す。なお、酵素活性の測定における標準基質とし
ては、フィルタ−ペ−パ−（Ｔｏｙｏ Ｒｏｓｈｉ Ｃ
ｏ．、ＦＰ−５Ｃ）、ＣＭＣ（和光純薬社製）および
Ａｖｉｃｅｌ（Ｍｅｒｃｈ Ｃｏ． ）を用いた。AKM-053
1 AKM-1511
RCH3
CH3 AO
CH2 CH2 O CH2 CH2
On
9 30
k 30
10
MW 18,000
16,000 The characteristics of modified cellulases obtained using each polymer were investigated. The results are shown below. In addition, as a standard substrate for measuring enzyme activity, filter paper (Toyo Roshi C
o. , FP-5C), CMC (manufactured by Wako Pure Chemical Industries, Ltd.) and
Avicel (Merch Co.) was used.
【００１９】ａ）化学修飾率および酵素活性修飾率は、
セルラ−ゼのアミノ酸残基中の未修飾アミノ基をＴＮＢ
Ｓ試薬で定量することにより決定した。ＡＫＭ−０５３
１ およびＡＫＭ−１５１１ で修飾した場合、最大修
飾率は５０％まで達した。a) Chemical modification rate and enzyme activity modification rate are:
The unmodified amino groups in the amino acid residues of cellulase are
It was determined by quantifying with S reagent. AKM-053
When modified with 1 and AKM-1511, the maximum modification rate reached up to 50%.
【００２０】ＡＫＭ−０５３１ およびＡＫＭ−１５１
１ で修飾したセルラ−ゼの酵素活性は、フィルタ−ペ
−パ−に対する活性で示すと、未修飾セルラ−ゼの活性
と比較して、修飾率２０％で約 ０．９５ 、同５０％
で０．８５〜０．９ であった。
これは、通常酵素の固定化で得られる残存活性よりも極
めて高い値である。比較のために無水マレイン酸で修飾
した場合には、修飾率は７０％まで達したが、残存活性
は ０．６程度まで低下した。AKM-0531 and AKM-151
The enzymatic activity of the cellulase modified with 1 is about 0.95 at a modification rate of 20%, and about 0.95 at a modification rate of 50%, compared to the activity of unmodified cellulase when expressed as activity on filter paper.
It was 0.85 to 0.9. This value is extremely higher than the residual activity normally obtained by immobilization of enzymes. For comparison, when modification was performed with maleic anhydride, the modification rate reached 70%, but the residual activity decreased to about 0.6.
【００２１】ｂ）熱安定性
ｐＨ ５．６、温度５０℃の条件下において、未修飾セ
ルラ−ゼの活性は９０時間後には初期活性の８０％まで
低下し、その後も経過時間に比例して活性低下が見られ
た。これに対して修飾セルラ−ゼの場合には、同条件に
おける９０時間後の活性は、３０％修飾の酵素で８５％
であり、５０％修飾の酵素では活性の低下は見られなか
った。すなわち、高分子で修飾することにより、熱安定
性を付与することができた。b) Thermostability Under conditions of pH 5.6 and temperature 50°C, the activity of unmodified cellulase decreases to 80% of its initial activity after 90 hours, and continues to increase in proportion to the elapsed time. A decrease in activity was observed. In contrast, in the case of modified cellulase, the activity after 90 hours under the same conditions was 85% for a 30% modified enzyme.
No decrease in activity was observed with the 50% modified enzyme. That is, thermal stability could be imparted by modifying with a polymer.
【００２２】ｃ）耐 ｐＨ 性
温度５０℃、ｐＨ ８．６で４８時間保存した後には、
未修飾セルラ−ゼはＣＭＣ、フィルタ−ペ−パ−のいず
れに対しても酵素活性が５０〜６０％に低下した。これ
に対して、修飾セルラ−ゼは、修飾率の増加と共に活性
の低下が少なくなる。上と同条件で保存した場合、４８
時間後の残存活性は、修飾率２０％で未修飾セルラ−ゼ
の １．２〜１．３ 倍、４０％で １．６倍、および
５０％で約 １．７倍と高い活性を示した。特に、修飾
率５０％の場合には、修飾直後の活性が４８時間後にも
ほとんど維持されていた。このように、高分子で修飾す
ることにより、耐 ｐＨ 安定性を付与することができ
る。c) pH resistance After storage for 48 hours at a temperature of 50°C and a pH of 8.6,
The enzyme activity of unmodified cellulase against both CMC and filter paper was reduced to 50-60%. In contrast, the activity of modified cellulases decreases less as the modification rate increases. If stored under the same conditions as above, 48
The residual activity after hours was 1.2 to 1.3 times that of unmodified cellulase at a modification rate of 20%, 1.6 times at 40%, and approximately 1.7 times at 50%. . In particular, when the modification rate was 50%, the activity immediately after modification was almost maintained even after 48 hours. In this way, by modifying with a polymer, pH stability can be imparted.
【００２３】ｄ）耐エタノ−ル性
セルラ−ゼはエタノ−ル共存下では活性低下を生じる。
例えば、 ５％エタノ−ル共存下では初期活性の４２％
、１０％エタノ−ル共存下では１５％程度まで低下して
しまう。
修飾セルラ−ゼはこの活性低下の割合を減ずることが可
能であり、 ５％エタノ−ル共存下で未修飾セルラ−ゼ
の約 １．５倍、１０％エタノ−ル共存下で ２倍以上
の酵素活性を示した。d) Ethanol-resistant cellulase has a decreased activity in the coexistence of ethanol. For example, in the presence of 5% ethanol, 42% of the initial activity
, it decreases to about 15% in the coexistence of 10% ethanol. Modified cellulase is able to reduce this rate of activity decline; it is about 1.5 times that of unmodified cellulase in the coexistence of 5% ethanol, and more than twice that of unmodified cellulase in the coexistence of 10% ethanol. It showed enzyme activity.
【００２４】ｅ）有機溶媒に対する溶解性と活性有機溶
媒としてアセトンを用いた場合を例にとると、未修飾セ
ルラ−ゼは、アセトン濃度５０％以上の水溶液では完全
に沈殿してしまい、溶液中の酵素活性は０となってしま
う。これに対して、修飾率４０％の修飾セルラ−ゼは、
アセトン濃度が８０％の水溶液でも完全に溶解し、溶液
中で酵素活性を維持する。すなわち、修飾セルラ−ゼは
有機溶媒に対する優れた溶解性を有しており、高濃度の
有機溶媒存在下においても酵素活性を示す。e) Solubility and activity in organic solvents Taking the case of using acetone as an organic solvent as an example, unmodified cellulase will completely precipitate in an aqueous solution with an acetone concentration of 50% or more; The enzyme activity of will be 0. On the other hand, modified cellulase with a modification rate of 40%,
It is completely dissolved even in an aqueous solution with an acetone concentration of 80%, and maintains enzyme activity in the solution. That is, the modified cellulase has excellent solubility in organic solvents and exhibits enzymatic activity even in the presence of high concentrations of organic solvents.
【００２５】このようなアセトン水溶液に対する溶解性
を利用して、未溶成分と修飾セルラ−ゼとを容易に分離
することができる。また、回収した修飾セルラ−ゼの溶
液部分を凍結乾燥して得た精製修飾セルラ−ゼは、場合
によっては、初期活性以上の活性を示すこともある。Utilizing such solubility in acetone aqueous solution, undissolved components and modified cellulase can be easily separated. In some cases, purified modified cellulase obtained by freeze-drying the recovered modified cellulase solution may exhibit an activity higher than the initial activity.
【００２６】以上のように、この発明による修飾セルラ
−ゼは、元の未修飾セルラ−ゼと比較して、耐熱性、耐
ｐＨ 性、耐アルコ−ル性、耐有機溶媒性等の実用的
見地から要求される諸性質が向上していることが明らか
である。As described above, the modified cellulase according to the present invention has practical advantages such as heat resistance, pH resistance, alcohol resistance, and organic solvent resistance compared to the original unmodified cellulase. It is clear that the required properties have improved from this point of view.
【００２７】実施例２
実施例１において調製した修飾セルラ−ゼを用いて、フ
ィルタ−ペ−パ−の糖化試験を行なった。Example 2 Using the modified cellulase prepared in Example 1, a filter paper saccharification test was conducted.
【００２８】ｐＨ ５．６、温度５０℃でフィルタ−ペ
−パ−を糖化させた場合には、反応初期には未修飾セル
ラ−ゼの方が糖転化率が高いものの、約３０時間後に転
化率が約３０％に達した時点で両者の差はなくなった。
その後、未修飾セルラ−ゼは転化速度が鈍り、９０時間
後の糖転化率が４０数％に止まった。一方、修飾セルラ
−ゼの転化速度はあまり低下せず、９０時間後には糖転
化率が５０数％と、未修飾セルラ−ゼの約 １．３倍と
なり、その後も両者の差は開く一方であった。[0028] When filter paper is saccharified at pH 5.6 and temperature 50°C, unmodified cellulase has a higher saccharide conversion rate at the beginning of the reaction, but the conversion rate decreases after about 30 hours. The difference between the two disappeared when the rate reached approximately 30%. Thereafter, the conversion rate of the unmodified cellulase slowed down, and the sugar conversion rate after 90 hours remained at about 40%. On the other hand, the conversion rate of the modified cellulase did not decrease much, and after 90 hours, the sugar conversion rate was about 50%, about 1.3 times that of the unmodified cellulase, and the difference between the two continued to widen. there were.
【００２９】このように、この発明による修飾セルラ−
ゼは、セルラ−ゼの実用性を高め、植物性バイオマスの
エネルギ−資源化など未利用有機資源の有効利用に好適
に用いることができる。[0029] Thus, the modified cellular according to the present invention
Cellulase enhances the practicality of cellulase and can be suitably used for the effective use of unused organic resources such as converting plant biomass into energy resources.
【００３０】[0030]
【発明の効果】以上のように、この発明の修飾セルラ−
ゼは、活性安定性、ｐＨ安定性、耐アルコ−ル性等の実
用的見地から要求される諸性質が向上している。したが
って、未修飾のセルラ−ゼでは使用不可能であった環境
で使用することが可能となり、その用途が拡大する。こ
のため、現在未利用の有機資源の有効利用等に有用であ
る。また、分離・回収を行ない易い形態であるため、容
易に再利用することができ、高価なセルラ−ゼを効率よ
く使用することができる。[Effects of the Invention] As described above, the modified cellular according to the present invention
The various properties required from a practical standpoint, such as activity stability, pH stability, and alcohol resistance, have been improved. Therefore, it can be used in environments where unmodified cellulase cannot be used, and its uses are expanded. Therefore, it is useful for effectively utilizing currently unused organic resources. Moreover, since it is in a form that is easy to separate and recover, it can be easily reused and expensive cellulase can be used efficiently.
Claims (2)
キル（メタ）アリルエ−テル・無水マレイン酸共重合体
で化学修飾されたセルラ−ゼ。1. A cellulase chemically modified with a polyoxyalkylene glycol alkyl (meth)allyl ether/maleic anhydride copolymer.
アルキル（メタ）アリルエ−テル・無水マレイン酸共重
合体が、下記化１に示される一般式（Ｉ）で表わされる
共重合体である請求項１記載の化学修飾セルラ−ゼ。 【化１】 （ここで、ＡＯはオキシアルキレン基、Ｒはアルキル基
をそれぞれ表わし、ｎは５〜５０、ｋは ５〜５０であ
る）2. The polyoxyalkylene glycol alkyl (meth)allyl ether/maleic anhydride copolymer is a copolymer represented by the general formula (I) shown in Formula 1 below. chemically modified cellulase. [Formula 1] (where AO represents an oxyalkylene group, R represents an alkyl group, n is 5 to 50, and k is 5 to 50)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3111959A JPH04341183A (en) | 1991-05-16 | 1991-05-16 | Chemically modified cellulase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3111959A JPH04341183A (en) | 1991-05-16 | 1991-05-16 | Chemically modified cellulase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04341183A true JPH04341183A (en) | 1992-11-27 |
| JPH0579309B2 JPH0579309B2 (en) | 1993-11-02 |
Family
ID=14574440
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3111959A Granted JPH04341183A (en) | 1991-05-16 | 1991-05-16 | Chemically modified cellulase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04341183A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07184673A (en) * | 1993-12-27 | 1995-07-25 | Nippon Oil & Fats Co Ltd | Production of phospholipid |
| JPH0856658A (en) * | 1994-08-29 | 1996-03-05 | Oriental Yeast Co Ltd | Stabilization of isocitric acid dehydrogenase |
| KR100242929B1 (en) * | 1997-09-25 | 2000-02-01 | 이원수 | Recycling method of waste paper by modified cellulase |
| EP0982396A2 (en) * | 1998-06-23 | 2000-03-01 | Korea Research Institute Of Chemical Technology | Modified enzyme and its modification process |
| JP2005206569A (en) * | 2003-03-24 | 2005-08-04 | Sankyo Co Ltd | Higher molecular modifier and medicinal composition |
| JP2006094811A (en) * | 2004-09-30 | 2006-04-13 | Tokyo Institute Of Technology | Modified laccase and method for treating aromatic compound |
-
1991
- 1991-05-16 JP JP3111959A patent/JPH04341183A/en active Granted
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07184673A (en) * | 1993-12-27 | 1995-07-25 | Nippon Oil & Fats Co Ltd | Production of phospholipid |
| JPH0856658A (en) * | 1994-08-29 | 1996-03-05 | Oriental Yeast Co Ltd | Stabilization of isocitric acid dehydrogenase |
| KR100242929B1 (en) * | 1997-09-25 | 2000-02-01 | 이원수 | Recycling method of waste paper by modified cellulase |
| EP0982396A2 (en) * | 1998-06-23 | 2000-03-01 | Korea Research Institute Of Chemical Technology | Modified enzyme and its modification process |
| EP0982396A3 (en) * | 1998-06-23 | 2001-02-28 | Korea Research Institute Of Chemical Technology | Modified enzyme and its modification process |
| JP2005206569A (en) * | 2003-03-24 | 2005-08-04 | Sankyo Co Ltd | Higher molecular modifier and medicinal composition |
| JP2006094811A (en) * | 2004-09-30 | 2006-04-13 | Tokyo Institute Of Technology | Modified laccase and method for treating aromatic compound |
| JP4636530B2 (en) * | 2004-09-30 | 2011-02-23 | 国立大学法人東京工業大学 | Process for treating aromatic compounds |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0579309B2 (en) | 1993-11-02 |
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