JPH02286082A - Method for separating and purifying chitin hydrolase - Google Patents
Method for separating and purifying chitin hydrolaseInfo
- Publication number
- JPH02286082A JPH02286082A JP11035289A JP11035289A JPH02286082A JP H02286082 A JPH02286082 A JP H02286082A JP 11035289 A JP11035289 A JP 11035289A JP 11035289 A JP11035289 A JP 11035289A JP H02286082 A JPH02286082 A JP H02286082A
- Authority
- JP
- Japan
- Prior art keywords
- chitin
- hydrolase
- enzyme
- exchange chromatography
- ion exchange
- 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.)
- Pending
Links
- 229920002101 Chitin Polymers 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims description 16
- 241000223259 Trichoderma Species 0.000 claims abstract description 14
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 12
- 230000002378 acidificating effect Effects 0.000 claims abstract description 10
- 150000001768 cations Chemical class 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims description 49
- 108090000790 Enzymes Proteins 0.000 claims description 49
- 230000000593 degrading effect Effects 0.000 claims description 19
- 239000007788 liquid Substances 0.000 abstract description 8
- 210000002421 cell wall Anatomy 0.000 abstract description 5
- 238000005119 centrifugation Methods 0.000 abstract description 5
- 210000004027 cell Anatomy 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 3
- 238000012258 culturing Methods 0.000 abstract description 3
- 238000001914 filtration Methods 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 238000012916 structural analysis Methods 0.000 abstract description 2
- 102000004157 Hydrolases Human genes 0.000 abstract 5
- 108090000604 Hydrolases Proteins 0.000 abstract 5
- 230000000694 effects Effects 0.000 description 21
- 102000012286 Chitinases Human genes 0.000 description 15
- 108010022172 Chitinases Proteins 0.000 description 15
- 239000000243 solution Substances 0.000 description 15
- 230000001794 chitinolytic effect Effects 0.000 description 13
- 239000000872 buffer Substances 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000000108 ultra-filtration Methods 0.000 description 5
- 108010055851 Acetylglucosaminidase Proteins 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 102000015962 Chitinase II Human genes 0.000 description 3
- 108050004344 Chitinase II Proteins 0.000 description 3
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 3
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 3
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 3
- 102100030122 Protein O-GlcNAcase Human genes 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 238000011033 desalting Methods 0.000 description 3
- 229950006780 n-acetylglucosamine Drugs 0.000 description 3
- 210000001938 protoplast Anatomy 0.000 description 3
- 238000005185 salting out Methods 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000238557 Decapoda Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000013024 dilution buffer Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 101710151413 Chitinase 1 Proteins 0.000 description 1
- 102100037328 Chitotriosidase-1 Human genes 0.000 description 1
- 101710132290 Chitotriosidase-1 Proteins 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 102100036495 Di-N-acetylchitobiase Human genes 0.000 description 1
- 101710107327 Endochitinase 1 Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108010000540 Hexosaminidases Proteins 0.000 description 1
- 102000002268 Hexosaminidases Human genes 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000002198 insoluble material Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
この発明はキチン分解酵素を分離精製する方法に関し、
更に詳しくは、コロイダルキチンを特異的に分解するキ
チン分解酵素を分離精製する方法に関するものである。This invention relates to a method for separating and purifying chitinolytic enzymes,
More specifically, the present invention relates to a method for isolating and purifying a chitin degrading enzyme that specifically decomposes colloidal chitin.
キチンはＮ−アセチルグルコサミンがβ−１゜４結合で
重合した多糖類であり、エビ、カニ等の甲殻類や昆虫等
の動物界に広く存在するとともに、カビ等の細胞壁構成
成分として微生物にも分布している。
かようなキチンのβ−１，４結合を切断するキチン分解
酵素であるキチナーゼは、動植物体内にもその存在が知
られているが、各種微生物由来のものも知られている。
本発明者らは、トリコデルマ（Ｔｒ！ｅｈｏｄｅｒＩＩ
ａ）属に属するＡｒｅ−Ｔｇ株の培養物からキチナーゼ
を採取できることを見出だし、既に特許出願を行った（
特願昭８３−１２３８４５号）。
キチ゛ナーゼの活性測定には、従来より種々の方法が提
案されている。通常は、基質としてコロイダルキチンを
使用し、濁度の減少あるいはＮ−アセチルグルコサミン
の生成量を測定する方法が採用されている。
しかしながら、濁度減少による活性１１＄１定法におい
てはキチナーゼ活性（液化型あるいはエンド型酵素活性
）が測定できるが、Ｎ−アセチルグルコサミン生成量に
よる活性１１−１定法では、共存するβ−Ｎ−アセチル
グルコサミニダーゼ活性（糖化型あるいはエキソ型酵素
活性）も同時に測定される。
キチン分解酵素の上記したごとき２つの活性を、ｉｉ定
する方法として、矢吹らは、コロイダルキチンの分解活
性（エンド型） ａｐｌ定法と、ｐ−ニトロフェニル−
β、Ｄ−Ｎ−アセチルグルコサミニドの分解活性（エキ
ソ型）ｌ−１定法を提案している（Ｊ、 Ｇｅｎ、＾ｐ
ｐ１． Ｍｌｃｒｏｂｉｏｌ、、 ８２．２５（１９１
１１Ｂ））。
本発明者らは、トリコデルマ属由来のキチン分解酵素に
ついて上記矢吹らの方法を用いて活性）Ｉ＋定を行った
ところ、コロイダルキチン分解活性を示す酵素と、ｐ−
ニトロフェニル−β。
Ｄ−Ｎ−アセチルグルコサミニド分解活性を示す酵素の
少なくとも２種類の酵素が存在することが判明した。
一般に、前者をキチナーゼ［Ｂ、Ｃ，３，２，１，目］
、後者をβ−Ｎ−アセチルグルコサミニダーゼまたはキ
トビアーゼ［Ｅ、ＣＪ、２．１．１７］と称するのが通
説となっているが、本明細書中ではキチナーゼを“キチ
ン分解酵素■” β−Ｎ−アセチルグルコサミニダーゼ
を“キチン分解酵素■”と略称する。Chitin is a polysaccharide made by polymerizing N-acetylglucosamine with β-1°4 bonds, and is widely present in the animal kingdom, including crustaceans such as shrimp and crabs, as well as insects, and is also used in microorganisms as a component of cell walls such as molds. It is distributed. Chitinases, which are chitinolytic enzymes that cleave the β-1,4 bonds of chitin, are known to exist in animals and plants, and are also known to be derived from various microorganisms. The present inventors have discovered that Trichoderma (Tr!ehoderII)
a) We discovered that chitinase can be collected from the culture of the Are-Tg strain belonging to the genus, and have already filed a patent application (
(Japanese Patent Application No. 123845/1983). Various methods have been proposed for measuring the activity of chitinase. Usually, a method is adopted in which colloidal chitin is used as a substrate and the reduction in turbidity or the amount of N-acetylglucosamine produced is measured. However, the activity 11-1 standard method based on turbidity reduction can measure chitinase activity (liquefied or endo-type enzyme activity), but the activity 11-1 standard method based on the amount of N-acetylglucosamine produced measures the amount of coexisting β-N-acetyl. Glucosaminidase activity (glycosylated or exo-type enzyme activity) is also measured at the same time. As a method for determining the above-mentioned two activities of chitin degrading enzymes, Yabuki et al.
β,D-N-acetylglucosaminide decomposition activity (exo type) l-1 standard method has been proposed (J, Gen, ^p
p1. Mlcrobiol, 82.25 (191
11B)). The present inventors performed activity) I+ determination on chitinolytic enzymes derived from the genus Trichoderma using the method of Yabuki et al., and found that enzymes exhibiting colloidal chitinolytic activity and p-
Nitrophenyl-β. It has been found that there are at least two types of enzymes that exhibit DN-acetylglucosaminide degrading activity. Generally, the former is chitinase [B, C, 3, 2, 1, order]
, the latter is generally referred to as β-N-acetylglucosaminidase or chitobiase [E, CJ, 2.1.17], but in this specification chitinase is referred to as “chitin-degrading enzyme ■” β-N- Acetylglucosaminidase is abbreviated as “chitin degrading enzyme ■”.
前述した特願昭６３−１２３６４５号においては、トリ
コデルマ属由来のキチン分□解酵素Ｉは、キチン質を用
いたアフィニティクロマトグラフィーやイオン交換クロ
マトグラフィーのごとき公知の方法によりキチン分解酵
素■と分離でき、５ＤＳ−ポリアクリルアミドゲル電気
泳動（以下“５ＯＳ−１）ＡＧＩ’：″と略記する）に
よって中−バンドとして観察されるところまで精製され
た旨記載されている。
しかしながら本発明者らのその後の研究により、精製さ
れたキチン分解酵素Ｉ中にも、微量のキチン分解酵素■
の活性が認められる場合があり、必ずしも精度よくキチ
ン分解酵素！と■の分離が行われていないことが判明し
た。
そこでこの発明は、トリコデルマ属の生産するキチン分
解酵素から、キチン分解酵素ＩＩを含まないキチン分解
酵素Ｉのみを採取し得る分離精製方法を提供することを
目的としてなされたものである。In the above-mentioned Japanese Patent Application No. 63-123645, chitin-degrading enzyme I derived from the genus Trichoderma can be separated from chitin-degrading enzyme II by known methods such as affinity chromatography using chitin or ion-exchange chromatography. , 5DS-polyacrylamide gel electrophoresis (hereinafter abbreviated as "5OS-1) AGI':") to the extent that it was observed as a medium band. However, subsequent research by the present inventors revealed that even in purified chitinase I, trace amounts of chitinase I
In some cases, the activity of chitinolytic enzymes is not necessarily accurate! It was found that the separation of and ■ was not carried out. Therefore, the present invention was made for the purpose of providing a method for separating and purifying only chitinase I, which does not contain chitinase II, from chitinases produced by the genus Trichoderma.
この発明によるキチン分解酵素の分離精製方法は、トリ
コデルマ属由来のキチン分解酵素！およびキチン分解酵
素ＩＩを含有する酵素液を強酸性陽イオン交換体を用い
るイオン交換クロマトグラフィーによって処理するもの
である。
キチン分解酵素ＩＩを含まないキチン分解酵素■は、こ
のイオン交換クロマトグラフィー処理の未吸着画分を採
取することにより得られる。
この発明の方法を実施するに際しては、先ずトリコデル
マ属由来のキチン分解酵素ｔａよびＩＩを含有する酵素
液を調製する。この酵素液は、例えば特願昭６３−１２
３６４５号に記載されているように、トリコデルマ属に
属するＡＦｅ−７８株を培養し、培養終了後、遠心分離
、濾過等で菌体を除去し、必要に応じて限外濾過濃縮、
硫安塩析等の処理を行うことにより得ることができる。
次いでこの酵素液を強酸性陽イオン交換体を用いるイオ
ン交換クロマトグラフィーによって処理する。強酸性陽
イオン交換体としては、例えば東ソー（株）製ｒｓＰ−
５ｐｗＪが好ましく使用できる。イオン交換クロマトグ
ラフィー操作は、通常のこの種クロマトグラフィー操作
と同様に、イオン交換体をカラムに充填し、このカラム
に酵素液を通液すればよい。１」的とするキチン分解酵
素Ｉは、未吸着画分として得ることができる。
かくして得られるキチン分解酵素■は、５ＤＳ−Ｐ Ａ
Ｇ ＩＥによる分析で単一であり、分子量的４０．０
００、至適ｐＨ４〜Ｂ、５（コロイダルキチンを基質と
した場合）、至適湯度３７℃（ｐＨ５，２、コロイダル
キチンを基質とした場合）であり、実質的なキチン分解
酵素■活性は認められない。The method for isolating and purifying a chitin degrading enzyme according to this invention is a chitin degrading enzyme derived from the Trichoderma genus! and an enzyme solution containing chitinolytic enzyme II is treated by ion exchange chromatography using a strongly acidic cation exchanger. Chitinase II, which does not contain Chitinase II, can be obtained by collecting the unadsorbed fraction from this ion exchange chromatography treatment. When carrying out the method of this invention, first, an enzyme solution containing chitin degrading enzymes ta and II derived from the genus Trichoderma is prepared. This enzyme solution can be used, for example, in Japanese Patent Application No. 63-12.
As described in No. 3645, AFe-78 strain belonging to the genus Trichoderma was cultured, and after the culture was completed, bacterial cells were removed by centrifugation, filtration, etc., and if necessary, ultrafiltration concentration,
It can be obtained by performing a treatment such as ammonium sulfate salting out. This enzyme solution is then treated by ion exchange chromatography using a strongly acidic cation exchanger. As the strongly acidic cation exchanger, for example, rsP- manufactured by Tosoh Corporation
5 pwJ can be preferably used. The ion exchange chromatography operation can be carried out in the same manner as in ordinary chromatography operations of this type, by filling a column with an ion exchanger and passing an enzyme solution through the column. The target chitinolytic enzyme I can be obtained as an unadsorbed fraction. The chitinolytic enzyme ■ thus obtained is 5DS-P A
Single by GIE analysis, molecular weight 40.0
00, optimal pH 4-B, 5 (when colloidal chitin is used as a substrate), optimal hot water temperature 37°C (pH 5.2, when colloidal chitin is used as a substrate), and the actual chitin degrading enzyme activity is unacceptable.
以下に実施例を挙げて、この発明の方法をさらに詳述す
る。
実施例１
（１）トリコデルマ 八［’６−Ｔｇ株の培養：ベブト
ン２．０ｇ 、酵母エキス０．５ｇ 、リン酸二水素カ
リウム １．Ｏｇ 、硫酸マグネシウム０．３ｇ１コロ
イダルキチン １．０ｇを水１ｇに溶解、懸濁し、ｐＨ
５，０に調整して培地を調製した。
培養は５Ｉジャーファメンタ−６基を使用し、１基につ
き培地１ｇを仕込み、各々に胞子懸濁液（２次種菌）
３００ ｍｌを植菌し、２５℃で培養した。植菌後、約
７２時間で培養を終了した。
（２）濾紙による濾過：
培養液を濾紙（Ｔｏｙｏ Ｎｏ、２ ）で濾過し、菌糸
をおおまかに除去した。
（３）限外濾過による濃縮、脱塩および不溶物の遠心分
離：
上記（２）で得られた培養濾液を限外濾過膜（分画分子
ｆｉｌｆ１．０００ ）で濃縮するとともに、限外濾過
の濃縮液の導電率が１００μｓ以下となるまで加水、脱
塩操作を繰り返した。この濃縮液に不溶物が認められた
ため、遠心分離（９，０００Ｇ）によって不溶物を除去
した。
（４）キチン・アフィニティ
上記（３）で得られた限外濾過濃縮液８４０１（総キチ
ナーゼ活性４Ｂ、５００υ）に、コロイダルキチン溶液
５５０ｇ （固形分含量６．５％）およびｐＨ５，２の
緩衝液（マツキルベイン緩衝液、以下同じ） １００ｇ
１を加え（導電率１．２ｇ＋８ ） 、５℃で１０分間
攪拌し、遠心分離（１２，０００Ｇ ）で沈殿を回収し
た。この沈殿に１０倍希釈緩衝液（ｐＨ５，２）を１，
０００ｓ＋ｌ加え（導電率１．４ｍｓ ）、ミキサーで
懸濁後、前記と同じ条件で沈殿を回収した。この沈殿に
１０倍希釈緩衝液（ｐＨ５，２）を２．０００ｍ１加え
、ミキサーで懸濁後、２５℃でインキュベーションを開
始した。２４時間後、インキュベーションを中止すると
とともに（総液ｆｆ１２．２００ｍ１ ） 、限外濾過
（分画分子ｊ１６．ｏｏｏ）よる濃縮および脱塩を行っ
た（総液量５３０ｍ１）。（５）硫安塩析、透析：上記
（４）で得られたキチン・アフィニティ処理液を濃縮す
るために、９５％硫安飽和による塩析を行なった後、透
析を行なった（液量７３１）。
（６）弱酸性イオン交換クロマトグラフィーによる精製
：
弱酸性陽イオン交換体（東ソー（株）製；１）ＥＡＢ−
６５０ｓ Ｔｏｙｏｐｅａｒｌ ）を充填したカラムに
、上記（５）で得られた濃縮液約７３−１を通液し、次
いテ２ｍｓ緩衝液（ｐＨ８，０）および２−８緩衝液（
ｐＨ４，０）を用いて、ｐＨグラジェントによる溶出を
行なった。
（７）強酸性イオン交換クロマトグラフィーによる精製
：
強酸性陽イオン交換体（東ソー（株）製；５Ｐ−５ｐν
）を充填したカラムに、上記（６）で得られた溶出液４
１を通液し、未吸着画分を採取した（２１１Ｓ緩衝液（
ｐＨ４，５）を使用）。
上記した各工程毎のキチン分解酵素Ｉの精製程度を第１
表に、５ＤＳ−ＰＡにＥによる分析結果を第１図に、そ
れぞれ示す。
なお、キチン分解酵素ｌおよび■の活性ｒｉ１１定は、
原則として矢吹らの方法（Ｙａｂｕｋｉ、Ｍ、ら。
Ｊ、Ｇｅｎ、 ＾ｐｐ１．Ｍｌｃｒｏｂｌｏ１．．３
２．２５ ＜１９８８））に従い、蛋白質の定量はＬ
ｏｖｒｙらの方法（Ｌｏｗｒｙ。
０、＋１．ら、」、旧ｏｆ、 Ｃｈｏｓ、、 １９３．
２８５ （１９５１） ）によった。また５ＤＳ−ＰＡ
ＧｒＪは、常法（Ｗｅｂｏｒ、に、および０ｓｂｏｒｎ
、Ｍ、、 Ｊ、旧ｏ１． Ｃｈｅｗ、、 ２４４．４４
０６（１９８９））に従った。
得られた精製キチン分解酵素Ｉは、第１図に示したよう
に５Ｏ８−ＰＡＧＥで単一であり、さらにその両分には
キチン分解酵素■活性は認められなかった。゛このＡＰ
８−Ｔｌ１株からのキチン分解酵素■の分子量は約４０
，０００であり、その比活性は約１１０ Ｕ ／Ｂ蛋白
質であった。
第１表：キチン分解酵素１の精製例
実施例２
実施例１における（６）の工程までの処理を行なって部
分精製した酵素液（キチン分解酵素ｌおよびＩＩを含み
、キチン分解酵素Ｉの活性は約１４０υ／ｍｇ）を用い
て以下の試験を実施した。
酵素液５１を強酸性陽イオン交換体５Ｐ−５ｐｖを充填
したカラムに通液し、未吸着画分を採取した（２ｓＳ緩
衝液（ｐＨ５，２）を使用）。
次いで、０．５Ｍ塩化ナトリウムを含む緩衝液で溶出を
行ない、２８０１■の吸光度でピークとじて検出された
両分も採取した。
強酸性イオン交換クロマトグラフィー処理した上記の未
吸着画分におけるキチン分解酵素Ｉの比活性は約１１０
１１７ａｇであり、この両分にはキチン分解酵素■の活
性は認められなかった。
８１’−５ｐｖ処理前の酵素液、未吸着画分および吸着
画分の５ＤＳ−ＰＡＧＥによる分析結果を第２図に示す
。第２図から、未吸着画分の酵素は単一であり、強酸性
イオン交換クロマトグラフィー処理によってキチン分解
酵素■が分離精製されていることがわかる。The method of the present invention will be explained in further detail with reference to Examples below. Example 1 (1) Cultivation of Trichoderma 8 ['6-Tg strain: 2.0 g of Bebuton, 0.5 g of yeast extract, potassium dihydrogen phosphate 1. Og, 0.3 g of magnesium sulfate 1.0 g of colloidal chitin was dissolved and suspended in 1 g of water, and the pH
A medium was prepared by adjusting the concentration to 5.0. For culturing, six 5I jar fermenters were used, each containing 1 g of culture medium, and a spore suspension (secondary inoculum) was added to each.
300 ml was inoculated and cultured at 25°C. Cultivation was completed approximately 72 hours after inoculation. (2) Filtration with filter paper: The culture solution was filtered with filter paper (Toyo No. 2) to roughly remove mycelia. (3) Concentration, desalting, and centrifugation of insoluble materials by ultrafiltration: The culture filtrate obtained in (2) above is concentrated with an ultrafiltration membrane (fraction molecule filf1.000), and the The operations of adding water and desalting were repeated until the conductivity of the concentrated solution became 100 μs or less. Since insoluble matter was observed in this concentrated solution, the insoluble matter was removed by centrifugation (9,000G). (4) Chitin affinity To the ultrafiltration concentrate 8401 (total chitinase activity 4B, 500υ) obtained in (3) above, add 550 g of colloidal chitin solution (solid content 6.5%) and a buffer solution of pH 5.2. (Matsukiruvain buffer, same below) 100g
1 (electrical conductivity: 1.2 g + 8), stirred at 5° C. for 10 minutes, and collected the precipitate by centrifugation (12,000 G). Add 10 times dilution buffer (pH 5,2) to this precipitate.
After adding 000 s+l (conductivity: 1.4 ms) and suspending with a mixer, the precipitate was collected under the same conditions as above. 2.000 ml of 10-fold dilution buffer (pH 5.2) was added to this precipitate, suspended in a mixer, and then incubation was started at 25°C. After 24 hours, incubation was stopped (total liquid volume ff12.200 ml), and concentration and desalting by ultrafiltration (fraction molecule j16.ooo) were carried out (total liquid volume 530 ml). (5) Ammonium sulfate salting out and dialysis: In order to concentrate the chitin affinity treated liquid obtained in (4) above, dialysis was performed after salting out with 95% ammonium sulfate saturation (liquid volume: 731). (6) Purification by weakly acidic ion exchange chromatography: Weakly acidic cation exchanger (manufactured by Tosoh Corporation; 1) EAB-
Approximately 73-1 of the concentrated solution obtained in step (5) above was passed through a column packed with 2ms buffer (pH 8,0) and 2-8 buffer (650s Toyopearl).
Elution was performed using a pH gradient (pH 4, 0). (7) Purification by strongly acidic ion exchange chromatography: Strongly acidic cation exchanger (manufactured by Tosoh Corporation; 5P-5pν
) into a column packed with eluate 4 obtained in (6) above.
1 was passed through the solution, and the unadsorbed fraction was collected (211S buffer (
pH 4,5)). The degree of purification of chitin degrading enzyme I for each step described above is determined as follows.
The results of analysis by E on 5DS-PA are shown in the table and in FIG. 1, respectively. In addition, the activity ri11 constant of chitinolytic enzyme l and ■ is as follows:
In principle, the method of Yabuki et al.
2.25 <1988)), protein quantification was performed using L
The method of ovry et al. (Lowry. 0, +1. et al., formerly of Chos, 193.
285 (1951)). Also 5DS-PA
GrJ is the conventional method (Webor, ni, and 0sborn
, M, , J, old o1. Chew,, 244.44
06 (1989)). As shown in FIG. 1, the obtained purified chitinolytic enzyme I was single in 5O8-PAGE, and chitinase I activity was not observed in both parts.゛This AP
8-The molecular weight of chitin degrading enzyme ■ from the Tl1 strain is approximately 40
,000, and its specific activity was approximately 110 U/B protein. Table 1: Purification example of chitin degrading enzyme 1 Example 2 Enzyme solution partially purified by processing up to step (6) in Example 1 (containing chitin degrading enzymes I and II, with activity of chitin degrading enzyme I) (approximately 140 υ/mg) was used to conduct the following test. The enzyme solution 51 was passed through a column packed with a strongly acidic cation exchanger 5P-5pv, and the unadsorbed fraction was collected (using 2sS buffer (pH 5, 2)). Next, elution was carried out with a buffer containing 0.5 M sodium chloride, and both fractions detected as a peak at absorbance of 2801 ■ were also collected. The specific activity of chitin degrading enzyme I in the above unadsorbed fraction treated with strong acid ion exchange chromatography is approximately 110.
117ag, and no chitin degrading enzyme (■) activity was observed in these two components. The results of 5DS-PAGE analysis of the enzyme solution, unadsorbed fraction, and adsorbed fraction before 81'-5pv treatment are shown in FIG. From FIG. 2, it can be seen that the unadsorbed fraction contained only a single enzyme, and that the chitin degrading enzyme ① was separated and purified by strong acid ion exchange chromatography treatment.
上述したところかられかるようにこの発明の方法によれ
ば、トリコデルマ属由来のキチン分解酵素から、キチン
をエンド型に切断するキチナーゼ（キチン分解酵素Ｉ）
を、エキソ型に切断するβ−Ｎ−アセチルグルコサミニ
ダーゼ（キチン分解酵素■）を含まない状態で分離精製
することができる。
トリコデルマ属の生産するキチン分解酵素は、従来から
プロトプラスト融合のためのプロトプラスト調製用の有
効な細胞壁溶解酵素として利用されているが、この発明
で得られるより一層精製されたキチン分解酵素１は、単
なるプロトプラスト調製用試薬としてだけでなく、細胞
壁の構造解析用試薬としての利用も可能となる。
さらに、細胞壁等からの有用物質の生産や、菌体内から
の有用物質の温和な抽出等にも利用でき、キチン分解酵
素の用途を広範囲に拡げることができる。As can be seen from the above, according to the method of the present invention, a chitinase (chitinase I) that cleaves chitin into an endo-type is obtained from a chitinase derived from the genus Trichoderma.
can be separated and purified without containing β-N-acetylglucosaminidase (chitinolytic enzyme ①) which cleaves it into exo form. Chitinolytic enzymes produced by the genus Trichoderma have been used as effective cell wall lytic enzymes for protoplast preparation for protoplast fusion, but the more purified chitinase 1 obtained in this invention is a simple It can be used not only as a reagent for protoplast preparation, but also as a reagent for cell wall structural analysis. Furthermore, it can be used for the production of useful substances from cell walls and the like, and for the gentle extraction of useful substances from microbial cells, thereby expanding the uses of chitinolytic enzymes over a wide range of areas.
第１図は実施例１における各工程毎の精製程度を示す５
Ｏ８−ＰＡＧＥ分析図、第２図は実施例２における５Ｐ
−５ｐｖ処理前の酵素液、未吸着画分および吸着画分の
５ＤＳ−ＰＡＧＥ分析図である。
なお図中の分子量マーカーとして使用した物質は次の通
りである。
分子量
１４．４００
２Ｇ、 ｔｏｏ
３０．０Ｇ０
４！１．０００
ｆ１７．ｏｏｏ
９４．０００Figure 1 shows the degree of purification in each step in Example 1.
O8-PAGE analysis diagram, Figure 2 is 5P in Example 2
-5DS-PAGE analysis diagram of the enzyme solution, unadsorbed fraction, and adsorbed fraction before treatment with 5 pv. The substances used as molecular weight markers in the figure are as follows. Molecular weight 14.400 2G, too 30.0G0 4!1.000 f17. ooo 94.000
Claims (1)
チン分解酵素IIを含有する酵素液を強酸性陽イオン交換
体を用いるイオン交換クロマトグラフィーによって処理
し、未吸着画分を採取することを特徴とするキチン分解
酵素１の分離精製方法。1. Chitin characterized by treating an enzyme solution containing chitin degrading enzyme I and chitin degrading enzyme II derived from the genus Trichoderma by ion exchange chromatography using a strongly acidic cation exchanger, and collecting the unadsorbed fraction. Method for separating and purifying degrading enzyme 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11035289A JPH02286082A (en) | 1989-04-28 | 1989-04-28 | Method for separating and purifying chitin hydrolase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11035289A JPH02286082A (en) | 1989-04-28 | 1989-04-28 | Method for separating and purifying chitin hydrolase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH02286082A true JPH02286082A (en) | 1990-11-26 |
Family
ID=14533590
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP11035289A Pending JPH02286082A (en) | 1989-04-28 | 1989-04-28 | Method for separating and purifying chitin hydrolase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02286082A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5173419A (en) * | 1991-06-17 | 1992-12-22 | Cornell Research Foundation, Inc. | Purified chitinases and use thereof |
| WO1994024271A1 (en) * | 1993-04-21 | 1994-10-27 | Cornell Research Foundation, Inc. | Nagase and glucosidase isolated from trichoderma harzianum and antifungal synergistic combinations of fungal cell wall degrading enzymes |
| US5378821A (en) * | 1991-06-17 | 1995-01-03 | Cornell Research Foundation, Inc. | Gene encoding for endochitinase |
| US6020540A (en) * | 1993-04-14 | 2000-02-01 | Cornell Research Foundation, Inc. | Gene encoding endochitinase |
| US6251390B1 (en) | 1991-06-17 | 2001-06-26 | Cornell Research Foundation, Inc. | Purified chitinases and use thereof |
-
1989
- 1989-04-28 JP JP11035289A patent/JPH02286082A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5173419A (en) * | 1991-06-17 | 1992-12-22 | Cornell Research Foundation, Inc. | Purified chitinases and use thereof |
| WO1992022314A1 (en) * | 1991-06-17 | 1992-12-23 | Cornell Research Foundation, Inc. | Purified chitinases and use thereof |
| US5378821A (en) * | 1991-06-17 | 1995-01-03 | Cornell Research Foundation, Inc. | Gene encoding for endochitinase |
| US6251390B1 (en) | 1991-06-17 | 2001-06-26 | Cornell Research Foundation, Inc. | Purified chitinases and use thereof |
| US5474926A (en) * | 1992-12-15 | 1995-12-12 | Cornell Research Foundation, Inc. | N-acetyl-β-glucosaminidase isolated from Trichoderma harzianum |
| US6020540A (en) * | 1993-04-14 | 2000-02-01 | Cornell Research Foundation, Inc. | Gene encoding endochitinase |
| WO1994024271A1 (en) * | 1993-04-21 | 1994-10-27 | Cornell Research Foundation, Inc. | Nagase and glucosidase isolated from trichoderma harzianum and antifungal synergistic combinations of fungal cell wall degrading enzymes |
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