JP2685226B2 - Method for separating chitinolytic enzyme and chitosan degrading enzyme - Google Patents
Method for separating chitinolytic enzyme and chitosan degrading enzymeInfo
- Publication number
- JP2685226B2 JP2685226B2 JP14051588A JP14051588A JP2685226B2 JP 2685226 B2 JP2685226 B2 JP 2685226B2 JP 14051588 A JP14051588 A JP 14051588A JP 14051588 A JP14051588 A JP 14051588A JP 2685226 B2 JP2685226 B2 JP 2685226B2
- Authority
- JP
- Japan
- Prior art keywords
- chitosan
- enzyme
- degrading enzyme
- ultrafiltration membrane
- chitin
- 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.)
- Expired - Lifetime
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- Enzymes And Modification Thereof (AREA)
Description
【発明の詳細な説明】 〈産業上の利用分野〉 本発明はカニ、エビ等の甲殻類の殻やオキアミ等に含
まれているキチンを分解する酵素、及び当該キチンを脱
アセチル化して得られるキトサンを分解する酵素を、そ
れらを産生する微生物の培養液から効率よく分離する方
法に関する。DETAILED DESCRIPTION OF THE INVENTION <Industrial field of application> The present invention can be obtained by degrading chitin, which is an enzyme that decomposes chitin contained in crustacean shells such as crab and shrimp and krill and the like. The present invention relates to a method for efficiently separating an enzyme that decomposes chitosan from a culture solution of a microorganism that produces them.
〈従来の技術〉 キチン及びキトサンは、それ自身フィルム、繊維、医
療材料、化粧品素材、各種酵素の担体、凝集剤等として
有用であり最近特に注目されているが、キチンを酵素分
解して得られるＮ−アセチルグリコサミンのモノマー及
びポリマー、キトサンを酵素分解して得られるグルコサ
ミンのモノマー及びポリマー等の分解物も極めて有用で
あり、例えば抗ガン剤や天然保存料等への適用を始めと
して、その利用方法が多くの分野で研究されつつあり、
一部は既に実用化されている。<Prior Art> Chitin and chitosan are themselves useful as films, fibers, medical materials, cosmetic materials, carriers for various enzymes, flocculants, etc., and have recently attracted particular attention, but they can be obtained by enzymatically degrading chitin. Degradation products of N-acetylglycosamine monomers and polymers, glucosamine monomers and polymers obtained by enzymatically degrading chitosan are also very useful, and for example, application to anticancer agents, natural preservatives, etc. How to use is being studied in many fields,
Some have already been put to practical use.
上述のようなキチン、キトサンの分解に使用されるキ
チン分解酵素及びキトサン分解酵素は、一般にこれらの
酵素を産生する微生物、例えばストレプトミセス属等の
放線菌、シュードモナス属及びビブリオ属等の細菌、ア
スペルギルス属等の糸状菌等を、カニ、エビ等の殻を基
質として培養することによって得られる。これらの酵素
は、前述のようなキチン分解物やキトサン分解物を生産
するための酵素製剤としてだけでなく、例えば植物病原
菌であるフザリウム菌等の増殖、生育を強く抑制する酵
素農薬として、あるいは微生物細胞壁中のキチンまたは
キトサンを分解する細菌壁融解酵素として、更に医療用
酵素剤等としても有用であることが確認されており、広
い用途と大きな需要が期待されている。Chitin as described above, chitin-degrading enzymes and chitosan-degrading enzymes used for degrading chitosan are generally microorganisms that produce these enzymes, for example, actinomycetes such as Streptomyces, bacteria such as Pseudomonas and Vibrio, Aspergillus. It can be obtained by culturing a filamentous fungus of the genus or the like using a shell of crab, shrimp or the like as a substrate. These enzymes are not only as an enzyme preparation for producing the above-mentioned chitin decomposition product and chitosan decomposition product, but also as an enzyme pesticide that strongly suppresses the growth and growth of, for example, the plant pathogen Fusarium bacterium, or a microorganism. It has been confirmed that it is useful as a bacterial wall-melting enzyme that decomposes chitin or chitosan in the cell wall, and also as a medical enzyme agent, and is expected to have wide applications and great demand.
しかし、このように有用なキチン分解酵素及びキトサ
ン分解酵素を産生する微生物及び当該微生物の培養方法
に関する研究は従来から多くなされているが、微生物の
培養液から前記酵素を分離あるいは抽出する方法に関す
る研究事例は非常に少なく特にこれらの酵素を工業的規
模で大量生産し得るような分離、抽出方法は見出されて
いない。However, much research has been done on microorganisms that produce such useful chitin-degrading enzymes and chitosan-degrading enzymes and methods for culturing the microorganisms, but studies on methods for separating or extracting the enzymes from the culture solution of the microorganisms have been conducted. Very few cases have been found, and in particular, no separation and extraction methods have been found that can mass-produce these enzymes on an industrial scale.
例えば、従来一般的に知られている抽出方法として
は、キチンをコロイド化したコロイダルキチンを、培養
液に加えてキチン分解酵素を当該コロイダルキチンに吸
着させて抽出する方法、同様にしてコロイド化したコロ
イダルキトサンにキトサン分解酵素を吸着させて抽出す
る方法がある。For example, as a generally known extraction method, colloidal chitin obtained by colloidal chitin is added to a culture solution to adsorb a chitin-degrading enzyme to the colloidal chitin and then extracted. There is a method in which colloidal chitosan is adsorbed with a chitosan degrading enzyme and then extracted.
〈発明が解決しようとする問題点〉 しかしながら、このような抽出方法は操作が極めて繁
雑であり、かつキチン分解酵素の収率が約30％程度、ま
たキトサン分解酵素の収率が約20％程度というように低
く、従って実験室規模での抽出方法としては適していて
も工業的規模での大量生産には不適であり、当該抽出方
法によって生産する場合には生産コストが著しく高くな
るという問題点を有していた。<Problems to be solved by the invention> However, such an extraction method is extremely complicated in operation, and the yield of chitin-degrading enzyme is about 30%, and the yield of chitosan-degrading enzyme is about 20%. Therefore, although it is suitable as an extraction method on a laboratory scale, it is unsuitable for mass production on an industrial scale, and when it is produced by the extraction method, the production cost becomes extremely high. Had.
上述のような事情から、従来ではキチン分解物及びキ
トサン分解物の応用研究あるいは上記酵素自体に関する
研究等に支障を来している面もあり、従ってこれらの酵
素を低コストで大量生産可能な分離、抽出方法の確立が
望まれていた。Due to the above-mentioned circumstances, there has been a problem that the conventional studies on the applied products of chitin degradation products and chitosan degradation products or the researches on the above-mentioned enzymes themselves have been hindered, and therefore these enzymes can be mass-produced at low cost. , Establishment of extraction method was desired.
本発明は上述のような背景のもとになされたものであ
り、キチン分解酵素とキトサン分解酵素とを含む培養液
から、これらの酵素を極めて簡単な操作で、かつ従来よ
り高い収率で分離し得る方法であって、工業的規模での
実施が可能な分離方法を提供することを目的とするもの
である。The present invention has been made based on the background as described above, and it is possible to separate these enzymes from a culture solution containing a chitin-degrading enzyme and a chitosan-degrading enzyme with a very simple operation and at a higher yield than ever before. It is an object of the present invention to provide a separation method which can be carried out on an industrial scale.
〈問題点を解決するための手段〉 本発明者等は、前記培養液からのキチン分解酵素及び
キトサン分解酵素の分離方法について鋭意研究を重ねた
結果、当該培養液を分画分子量の異なる複数の限外濾過
膜で順次処理するという極めて簡単な操作で前記酵素を
効率よく分離し得ることを見出し、当該知見に基づいて
本発明を成すに至った。<Means for Solving Problems> The inventors of the present invention have conducted extensive studies on a method for separating a chitin-degrading enzyme and a chitosan-degrading enzyme from the culture solution, and as a result, the culture solution was separated into a plurality of fractionated molecular weights different from each other. It was found that the enzyme can be efficiently separated by an extremely simple operation of sequentially treating with an ultrafiltration membrane, and the present invention has been completed based on this finding.
すなわち、本発明はキチン分解酵素とキトサン分解酵
素とを含む培養液を、先ず分画分子量20万〜50万の限外
濾過膜で処理して前記酵素類をほとんど含まない、前記
酵素類を産生する微生物菌体の濃縮液と、前記酵素類及
びキチン、キトサンの分解物を含む透過液とに分離し、
次いで当該透過液を分画分子量５万〜10万の限外濾過膜
で処理してキチン分解酵素の濃縮液とキトサン分解酵素
及び前記分解物を含む透過液とに分離し、更に当該透過
液を分画分子量１万〜３万の限外濾過膜で処理してキト
サン分解酵素の濃縮液と前記分解物を含む透過液とに分
離することを特徴とするものである。That is, the present invention, a culture solution containing a chitin-degrading enzyme and a chitosan-degrading enzyme is first treated with an ultrafiltration membrane having a cut-off molecular weight of 200,000 to 500,000 to produce the enzyme containing almost no enzyme. Separated into a concentrated liquid of microbial cells to do, a permeate containing the above enzymes and chitin, a decomposition product of chitosan,
Then, the permeate is treated with an ultrafiltration membrane having a molecular weight cut off of 50,000 to 100,000 to separate it into a concentrated solution of chitin-degrading enzyme and a permeate containing chitosan-degrading enzyme and the above-mentioned decomposed product. It is characterized in that it is treated with an ultrafiltration membrane having a molecular weight cut-off of 10,000 to 30,000 to separate it into a concentrated solution of chitosan-degrading enzyme and a permeate containing the above-mentioned degradation product.
〈作用〉 以下に本発明を図面を用いて詳細に説明する。<Operation> Hereinafter, the present invention will be described in detail with reference to the drawings.
例えば、カニ、エビ等の殻を基質としてキチン分解酵
素を産生する微生物とキトサン分解酵素を産生する微生
物とを培養した培養液からキチン分解酵素及びキトサン
分解酵素を分離する場合には、当該培養液を予め遠心分
離等の手段で前処理して培養液中の殻や高分子タンパク
質等の比較的粗大な懸濁物を除去しておき、これらを除
去した培養液を図面に示したようなフローにしたがって
処理する。For example, in the case of separating the chitin-degrading enzyme and the chitosan-degrading enzyme from the culture medium in which the microorganism producing the chitin-degrading enzyme and the microorganism producing the chitosan-degrading enzyme are cultivated using the shell of crab, shrimp, etc. Was pre-treated with a means such as centrifugation to remove relatively coarse suspensions such as shells and high molecular proteins in the culture solution, and the culture solution from which these were removed was subjected to the flow shown in the drawing. Process according to.
すなわち、カニ、エビ等の殻や高分子タンパク質等を
除去した培養液を原液槽１に一旦貯留し、しかる後に当
該培養液を分画分子量20万〜50万の範囲の限外濾過膜２
を装着した第１の限外濾過膜装置３に送給して処理す
る。当該処理においては、培養液中の微生物菌体やキチ
ン、キトサン等の極めて大きな分子のみを限外濾過膜２
の膜面で阻止することが出来、これらの物質を含まない
透過液４を得るとともに前記阻止した物質を濃縮した非
透過液５を原液槽１に循環する。得られる透過液４中に
は限外濾過膜２を透過したキチン分解酵素、キトサン分
解酵素及びキチン、キトサンそれぞれの分解物が含まれ
ているので、これを後段の中間槽６に導いて一旦貯留
し、次工程の限外濾過膜処理に供する。That is, the culture solution from which shells such as crabs and shrimp and high molecular proteins have been removed is temporarily stored in the stock solution tank 1, and then the culture solution is subjected to an ultrafiltration membrane 2 having a molecular weight cut-off of 200,000 to 500,000.
Is fed to the first ultrafiltration membrane device 3 to which it is attached for processing. In the treatment, only ultra-large molecules such as microbial cells and chitin and chitosan in the culture solution are used in the ultrafiltration membrane 2
The permeated liquid 4 containing no such substances can be obtained, and the non-permeated liquid 5 in which the blocked substances are concentrated is circulated to the stock solution tank 1. Since the permeated liquid 4 thus obtained contains chitin-degrading enzymes, chitosan-degrading enzymes and decomposed products of chitin and chitosan, which have permeated the ultrafiltration membrane 2, they are introduced to the intermediate tank 6 in the subsequent stage and temporarily stored. And subjected to ultrafiltration membrane treatment in the next step.
このような、透過液４を取り出しながらの循環処理に
より、原液槽１内には前記微生物菌体等が濃縮されると
ともに当該槽１内の溶液中にはキチン分解酵素及びキト
サン分解酵素がほとんど存在しなくなるので、その時点
で当該処理を停止する。最終的に原液槽１内に残留した
濃縮液は、例えば前述のような微生物培養に再び使用す
る。By the circulation processing while taking out the permeated liquid 4, the microbial cells and the like are concentrated in the stock solution tank 1, and the chitin-degrading enzyme and the chitosan-degrading enzyme are almost present in the solution in the tank 1. Since it will not be done, the process is stopped at that point. The concentrated liquid that finally remains in the stock solution tank 1 is reused, for example, in the above-described microbial culture.
なお、上述のようないわゆるバッチ処理においては、
処理を続行するにしたがって原液槽１内の溶液の濃度が
高くなり、限外濾過膜装置３の透過液量が少なくなった
り、あるいは微生物菌体等の粒子径の大きな不純物が透
過液側に漏出し易くなったりすることがあるが、このよ
うな障害をなくすために、原液槽１内に水を一時的に、
あるいは連続的に加えて原液槽１内の溶液を希釈しなが
ら処理を行うようにしてもよい。このような操作を行う
と、最終的に得られる濃縮液あるいは透過液が希釈され
ることとなるが、その反面原液槽１内の酵素類を透過液
側に効果的に「洗い出す」ことが出来、最終的に原液槽
１内に残留する酵素類の量を著しく少なくすることが出
来る。当該操作は以後の限外濾過膜処理においても適用
することが出来るのは勿論である。In the so-called batch processing as described above,
As the treatment is continued, the concentration of the solution in the stock solution tank 1 increases, the amount of permeate in the ultrafiltration membrane device 3 decreases, or impurities such as microbial cells having a large particle size leak to the permeate side. However, in order to eliminate such obstacles, water is temporarily stored in the stock solution tank 1,
Alternatively, the treatment may be carried out while continuously adding and diluting the solution in the stock solution tank 1. When such an operation is performed, the finally obtained concentrated liquid or permeate is diluted, but on the other hand, the enzymes in the stock solution tank 1 can be effectively “washed out” to the permeate side. Finally, the amount of enzymes remaining in the stock solution tank 1 can be significantly reduced. Of course, the operation can be applied to the subsequent ultrafiltration membrane treatment.
ここで、第１の限外濾過膜装置３に使用する限外濾過
膜２の分画分子量を20万〜50万の範囲としたのは、分画
分子量が50万を越える限外濾過膜を使用したのでは透過
液側に微生物菌体等が漏出し、最終的に得られるキチン
分解酵素やキトサン分解酵素の純度が低下するので好ま
しくなく、また分画分子量が20万未満の限外濾過膜を使
用したのでは、膜によりキチン分解酵素及びキトサン分
解酵素の一部が阻止されて濃縮液側に移行し、これらの
酵素の歩留まりが低下するので好ましくないからであ
る。Here, the reason that the molecular weight cut-off of the ultrafiltration membrane 2 used in the first ultrafiltration membrane device 3 is in the range of 200,000 to 500,000 is that an ultrafiltration membrane having a molecular weight cut-off of more than 500,000 is used. If used, it is not preferable because microbial cells leak to the permeate side and the purity of the finally obtained chitin-degrading enzyme or chitosan-degrading enzyme decreases, and the molecular weight cutoff is less than 200,000. This is because the use of is not preferable because a part of the chitin-degrading enzyme and the chitosan-degrading enzyme is blocked by the membrane and migrates to the concentrated liquid side, and the yield of these enzymes decreases.
次いで、中間槽６に貯留した透過液４を、分画分子量
５万〜10万の範囲の限外濾過膜７を装着した第２の限外
濾過膜装置８に送給して処理する。当該処理において
は、第１の限外濾過膜装置３の透過液４中に含まれてい
る、分子量約11万のキチナーゼ及びキトビアーゼ等のキ
チン分解酵素を限外濾過膜７の膜面で阻止することが出
来、当該キチン分解酵素を含まない透過液９を得るとと
もにキチン分解酵素を濃縮した非透過液10を前記中間槽
６に循環する。得られる透過液９中には限外濾過膜７を
透過したキトサン分解酵素及びキチン、キトサンそれぞ
れの分解物が含まれているので、これを後段の中間槽11
に導いて一旦貯留し、次工程の限外濾過膜処理に供す
る。Then, the permeated liquid 4 stored in the intermediate tank 6 is fed to the second ultrafiltration membrane device 8 equipped with the ultrafiltration membrane 7 having a molecular weight cut off of 50,000 to 100,000 for processing. In the treatment, a chitin-degrading enzyme such as chitinase and chitobiase having a molecular weight of about 110,000 contained in the permeate 4 of the first ultrafiltration membrane device 3 is blocked on the membrane surface of the ultrafiltration membrane 7. A permeate 9 containing no chitin-degrading enzyme is obtained, and a non-permeate 10 containing chitin-degrading enzyme is circulated to the intermediate tank 6. Since the permeated liquid 9 thus obtained contains the chitosan-degrading enzyme and the decomposed products of chitin and chitosan that have permeated through the ultrafiltration membrane 7, this is passed through the intermediate tank 11 at the subsequent stage.
To be stored once and used for the ultrafiltration membrane treatment in the next step.
このような循環処理により、中間槽６内にキチン分解
酵素を高濃度かつ高純度に含有する濃縮液を得ることが
出来るので適当な時期に当該処理を停止し、濃縮液を槽
６外に取り出す。当該濃縮液は、これをそのままキチン
分解酵素溶液として使用することも出来るし、あるいは
当該濃縮液から凍結乾燥等の手段によって更に精製され
た粉末状酵素を得ることも出来る。By such a circulation treatment, a concentrated liquid containing a chitin-degrading enzyme in a high concentration and high purity can be obtained in the intermediate tank 6, so the treatment is stopped at an appropriate time and the concentrated liquid is taken out of the tank 6. . The concentrated solution can be used as it is as a chitinolytic enzyme solution, or a powdery enzyme further purified from the concentrated solution by a means such as freeze-drying can be obtained.
ここで、第２の限外濾過膜装置８に使用する限外濾過
膜２の分画分子量を５万〜10万の範囲としたのは、分画
分子量10万を超える限外濾過膜を使用したのでは透過液
側にキチン分解酵素の一部が漏出し、次工程で得られる
キトサン分解酵素の純度を低下させることとなるので好
ましくなく、また分画分子量が５万未満の限外濾過膜を
使用したのでは、膜によりキトサン分解酵素の一部が阻
止されて濃縮液側に移行し、得られるキチン分解酵素溶
液の純度を低下させて好ましくないからである。Here, the reason that the molecular weight cutoff of the ultrafiltration membrane 2 used in the second ultrafiltration membrane device 8 is in the range of 50,000 to 100,000 is that an ultrafiltration membrane having a molecular weight cutoff of more than 100,000 is used. This is not preferable because a part of the chitin-degrading enzyme leaks to the permeate side and the purity of the chitosan-degrading enzyme obtained in the next step is reduced, which is not preferable, and the molecular weight cutoff is less than 50,000. This is because the use of is not preferable because a part of the chitosan-degrading enzyme is blocked by the membrane and migrates to the concentrated liquid side, which reduces the purity of the resulting chitin-degrading enzyme solution.
更に、中間槽11に貯留した透過液９を、分画分子量１
万〜３万の範囲の限外濾過膜12を装着した第３の限外濾
過膜装置13に送給して処理する。当該処理においては、
第２の限外濾過膜装置８の透過液９中に含まれているキ
トサン分解酵素（分子量約４万のキトサナーゼ）を限外
濾過膜12の膜面で阻止することが出来、当該キトサン分
解酵素を含まない透過液14を得るとともにキトサン分解
酵素を濃縮した非透過液15を前記中間槽11に循環する。Further, the permeated liquid 9 stored in the intermediate tank 11 is treated with a molecular weight cut-off of 1
It is fed to the third ultrafiltration membrane device 13 equipped with the ultrafiltration membrane 12 in the range of 10,000 to 30,000 for processing. In the process,
The chitosan degrading enzyme (chitosanase having a molecular weight of about 40,000) contained in the permeate 9 of the second ultrafiltration membrane device 8 can be blocked by the membrane surface of the ultrafiltration membrane 12, and the chitosan degrading enzyme And a non-permeate 15 in which the chitosan-degrading enzyme is concentrated is circulated in the intermediate tank 11.
このような循環処理により、中間槽11内にキトサン分
解酵素を高濃度かつ高純度に含む濃縮液を得ることが出
来るので適当な時期に当該処理を停止し、中間槽11内の
濃縮液をキトサン分解酵素溶液として取り出す。当該溶
液も、前述したキトサン分解酵素溶液の場合と同様に、
これをそのま使用することが出来るし、あるいは凍結乾
燥等によって当該濃縮液から更に精製された粉末状酵素
を得ることも出来る。なお、第３の限外濾過膜装置13の
透過液14中には、キチンの分解物であるＮ−アセチルグ
ルコサミンのモノマー及びポリマー、キトサンの分解物
であるアセチルグルコサミンのモノマー及びポリマー
等、非常に有用な物質が含まれているので、当該透過液
14も回収使用することが出来る。By such a circulation treatment, a concentrated liquid containing a chitosan degrading enzyme in a high concentration and a high purity can be obtained in the intermediate tank 11, so the treatment is stopped at an appropriate time, and the concentrated liquid in the intermediate tank 11 is treated with chitosan. It is taken out as a decomposing enzyme solution. The solution is also similar to the case of the chitosan-degrading enzyme solution described above,
This can be used as it is, or a powdery enzyme further purified from the concentrate can be obtained by freeze-drying or the like. In the permeate 14 of the third ultrafiltration membrane device 13, monomers and polymers of N-acetylglucosamine which is a decomposition product of chitin, monomers and polymers of acetylglucosamine which is a decomposition product of chitosan, etc. The permeated liquid contains useful substances.
14 can also be collected and used.
ここで、第３の限外濾過膜装置13に使用する限外濾過
膜12の分画分子量を１万〜３万の範囲としたのは、分画
分子量が３万を超える限外濾過膜を使用したのでは透過
液側にキトサン分解酵素が漏出し、当該酵素の歩留まり
が低下するので好ましくなく、また分画分子量が１万未
満の限外濾過膜を使用したのでは、膜により前述のよう
な分解物の一部が阻止されて濃縮液側に移行し、得られ
るキトサン分解酵素溶液の純度を低下させて好ましくな
いからである。Here, the reason that the molecular weight cutoff of the ultrafiltration membrane 12 used in the third ultrafiltration membrane device 13 is in the range of 10,000 to 30,000 is that the ultrafiltration membrane having a molecular weight cutoff of more than 30,000 is used. If it is used, it is not preferable because the chitosan-degrading enzyme leaks to the permeate side and the yield of the enzyme decreases, and if an ultrafiltration membrane with a molecular weight cut-off of less than 10,000 is used, it may be different depending on the membrane as described above. This is because some of the decomposition products are blocked and migrate to the concentrated liquid side, which reduces the purity of the obtained chitosan-degrading enzyme solution, which is not preferable.
なお、以上の操作は、操作中における各酵素の失活を
極力少なくするために、液温５〜10℃程度の低温下で行
うのが望ましい。The above operation is preferably performed at a low temperature of about 5 to 10 ° C. in order to minimize the inactivation of each enzyme during the operation.
また、上述の説明では理解を容易にするために各限外
濾過膜処理を完全なバッチ処理として説明したが、例え
ば第１の限外濾過膜装置による処理を行いながら、第２
の限外濾過膜装置で第１の限外濾過膜装置の透過液の処
理を行うようにしてもよいことは言うまでもないことで
ある。Further, in the above description, each ultrafiltration membrane treatment is described as a complete batch treatment for easy understanding, but, for example, while performing the treatment by the first ultrafiltration membrane device,
It goes without saying that the ultrafiltration membrane device may be used to process the permeate of the first ultrafiltration membrane device.
本発明に適用出来る各限外濾過膜は、それぞれ前述し
たような範囲の分画分子量の限外濾過膜であればいかな
るものでもよく、形状、材質等は問わない。Each ultrafiltration membrane applicable to the present invention may be any ultrafiltration membrane having a molecular weight cutoff within the above-mentioned range, and its shape, material and the like are not limited.
〈効果〉 本発明によれば、キチン分解酵素とキトサン分解酵素
とを含む培養液を、分画分子量の異なる複数の限外濾過
膜を用いて順次処理するという極めて簡単な操作でキチ
ン分解酵素とキトサン分解酵素とを各々単独で分離する
ことが出来、しかも従来法より高い収率で得ることが出
来る。従って、本発明方法により、これらの酵素の工業
的規模での大量生産が可能となり、これらの酵素を従来
よりはるかに安価に生産することが可能となる。また、
大量生産が可能になることからこれらの酵素の入手も容
易となり、よってキチンやキトサンの分解物の応用研究
や酵素自体の研究がより活発化されることが期待され、
本発明が産業の発展に寄与するところ極めて大である。<Effect> According to the present invention, a culture solution containing a chitin-degrading enzyme and a chitosan-degrading enzyme is treated with a chitin-degrading enzyme by an extremely simple operation of sequentially treating using a plurality of ultrafiltration membranes having different cutoff molecular weights. The chitosan-degrading enzyme can be separately isolated, and the yield can be higher than that of the conventional method. Therefore, the method of the present invention enables large-scale production of these enzymes on an industrial scale, and makes it possible to produce these enzymes at a much lower cost than before. Also,
Since it becomes possible to mass-produce these enzymes, it will be easier to obtain these enzymes, and it is expected that the applied research on the degradation products of chitin and chitosan and the research on the enzymes themselves will become more active.
The present invention greatly contributes to industrial development.
更に、本発明においては前記酵素類を分離した後に、
Ｎ−グルコサミンやグルコサミン等の有用物質を多く含
み、その他の不純物を余り含まない溶液を得ることが出
来、当該溶液もまた有効に活用し得るという利点を有す
る。Furthermore, in the present invention, after separating the enzymes,
There is an advantage that a solution containing a lot of useful substances such as N-glucosamine and glucosamine and containing little other impurities can be obtained, and the solution can also be effectively used.
〈実施例〉 以下に本発明の実施例を説明する。<Examples> Examples of the present invention will be described below.
キチン分解酵素を産生する微生物とキトサン分解酵素
を産生する微生物との混合微生物系（フラボバクテリウ
ム属１株、シュードモナス属１株及び未同定２株の混合
系）を、リン酸塩を含む脱Caカニ殻粉末を唯一の基質と
する培地で、常法により30℃にて10日間培養し、キチン
分解酵素とキトサン分解酵素とを含む培養液を得た。得
られた培養液を遠心分離機で処理してカニ殻、高分子タ
ンパク質等の比較的粗大な懸濁物を除去し、これを図面
に示したようなフローに従って順次処理した。使用した
限外濾過膜装置は第１〜第３のいずれもロミコン社製の
もので、それぞれ内径1.1mm、外径1.9mmのホローファイ
バー形限外濾過膜を多数本集束して外径２インチのカー
トリッジ内に充填した構造のものであり、膜面積はいず
れも0.46m2である。A mixed microbial system of a microorganism producing a chitinolytic enzyme and a microorganism producing a chitosan degrading enzyme (a mixed system of 1 Flavobacterium genus strain, 1 Pseudomonas strain and 2 unidentified strains) The culture medium containing crab shell powder as the only substrate was cultured at 30 ° C. for 10 days by a conventional method to obtain a culture solution containing a chitin-degrading enzyme and a chitosan-degrading enzyme. The obtained culture broth was treated with a centrifuge to remove relatively coarse suspensions of crab shells, high-molecular proteins, etc., which were sequentially treated according to the flow shown in the drawing. The ultrafiltration membrane devices used were all manufactured by Romicon Co., Ltd., and a large number of hollow fiber type ultrafiltration membranes each having an inner diameter of 1.1 mm and an outer diameter of 1.9 mm were bundled to form an outer diameter of 2 inches. Each of the membranes has a structure of 0.46 m 2 in which the membrane area is 0.46 m 2 .
また、使用した限外濾過膜は、第１の限外濾過膜装置
のそれが分画分子量50万のもの（膜材質ポリスルホ
ン）、第２の限外濾過膜装置のそれが分画分子量５万の
もの（膜材質親水性ポリマー）、また第３の限外濾過膜
装置のそれは分画分子量１万のもの（膜材質親水性ポリ
マー）である。The ultrafiltration membrane used had a molecular weight cutoff of 500,000 in the first ultrafiltration membrane device (polysulfone membrane material) and a molecular weight cutoff of 50,000 in the second ultrafiltration membrane device. (Membrane material hydrophilic polymer) and that of the third ultrafiltration membrane device have a cut-off molecular weight of 10,000 (membrane material hydrophilic polymer).
また、処理条件は液温10℃、各限外濾過膜装置の操作
圧力をいずれも入口圧1.75kg/cm2、出口圧1.0kg/cm2、
また非透過液の循環流量を900l/Hとして行った。なお、
この時の透過液の流量は、第１の限外濾過膜装置のそれ
が約18.4l/H、第２の限外濾過膜装置のそれが約13.8kl/
H、第３の限外濾過膜装置のそれが約32.2l/Hであった。The processing conditions were solution temperature 10 ° C., the ultrafiltration both the operating pressure of the membrane device inlet pressure 1.75 kg / cm 2, the outlet pressure of 1.0 kg / cm 2,
Further, the circulation flow rate of the non-permeated liquid was set to 900 l / H. In addition,
The flow rate of the permeate at this time is about 18.4 l / H for the first ultrafiltration membrane device and about 13.8 kl / H for the second ultrafiltration membrane device.
H, that of the third ultrafiltration membrane device was about 32.2 l / H.
以上のような条件のもとに、培養液40lを先ず第１の
限外濾過膜装置で処理して5lの濃縮液と35lの透過液と
を得、次いで当該35lの透過液を第２の限外濾過膜装置
で処理してキチン分解酵素を濃縮した濃縮液5lと透過液
30lとを得た。第２の限外濾過膜装置により処理によっ
て得られた濃縮液中のキチン分解酵素活性を測定し、当
該酵素の収率を求めたところ約40％であった。Under the above conditions, 40 l of the culture solution was first treated with the first ultrafiltration membrane device to obtain 5 l of concentrated solution and 35 l of permeate, and then 35 l of the permeated solution was treated with the second permeate. Concentrated liquid 5 l and permeated liquid in which chitin-degrading enzyme was concentrated by processing with an ultrafiltration membrane device
Got with 30l. The chitin-degrading enzyme activity in the concentrated solution obtained by the treatment with the second ultrafiltration membrane device was measured, and the yield of the enzyme was determined to be about 40%.
更に前記30lの透過液を第３の限外濾過膜装置で処理
してキトサン分解酵素を濃縮した濃縮液5lと透過液25l
とを得た。得られた濃縮液中のキトサン分解酵素活性を
測定し、当該酵素の収率を求めたところ約35％であっ
た。Further, 30 l of the permeate was treated with a third ultrafiltration membrane device to concentrate chitosan degrading enzyme 5 l and permeate 25 l
And got. The activity of the chitosan-degrading enzyme in the obtained concentrated liquid was measured, and the yield of the enzyme was determined to be about 35%.
図面は本発明の実施態様の一例を示すフローの説明図で
ある。 １……原液槽、２……限外濾過膜 ３……第１の限外濾過膜装置 ４……透過液、５……非透過液 ６……中間槽、７……限外濾過膜 ８……第２の限外濾過膜装置 ９……透過液、10……非透過液 11……中間槽、12……限外濾過膜 13……第３の限外濾過膜装置 14……透過液、15……非透過液The drawing is an explanatory diagram of a flow showing an example of an embodiment of the present invention. 1 ... stock solution tank, 2 ... ultrafiltration membrane 3 ... 1st ultrafiltration membrane device 4 ... permeate, 5 ... non-permeate liquid 6 ... intermediate tank, 7 ... ultrafiltration membrane 8 ...... Second ultrafiltration membrane device 9 …… Permeate, 10 …… Non-permeate 11 …… Intermediate tank, 12 …… Ultrafiltration membrane 13 …… Third ultrafiltration membrane device 14 …… Permeation Liquid, 15 ... Impermeable liquid
Claims (1)
む培養液を、先ず分画分子量20万〜50万の限外濾過膜で
処理して前記酵素類をほとんど含まない、前記酵素類を
産生する微生物菌体の濃縮液と、前記酵素類及びキチ
ン、キトサンの分解物を含む透過液とに分離し、次いで
当該透過液を分画分子量５万〜10万の限外濾過膜で処理
してキチン分解酵素の濃縮液と、キトサン分解酵素及び
前記分解物を含む透過液とに分離し、更に当該透過液を
分画分子量１万〜３万の限外濾過膜で処理してキトサン
分解酵素の濃縮液と、前記分解物を含む透過液とに分離
することを特徴とするキチン分解酵素及びキトサン分解
酵素の分離方法。1. A culture broth containing a chitin-degrading enzyme and a chitosan-degrading enzyme is first treated with an ultrafiltration membrane having a cut-off molecular weight of 200,000 to 500,000 to produce the enzyme containing almost no enzyme. And a permeate containing the above-mentioned enzymes and a decomposition product of chitin and chitosan, and the permeate is treated with an ultrafiltration membrane having a molecular weight cut off of 50,000 to 100,000. The chitolytic enzyme is concentrated and the permeated liquid containing the chitosan-degrading enzyme and the decomposition product is separated, and the permeated liquid is further treated with an ultrafiltration membrane having a cut-off molecular weight of 10,000 to 30,000 to obtain a chitosan-degrading enzyme. A method for separating a chitin-degrading enzyme and a chitosan-degrading enzyme, which comprises separating a concentrated solution and a permeate containing the above-mentioned decomposed product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14051588A JP2685226B2 (en) | 1988-06-09 | 1988-06-09 | Method for separating chitinolytic enzyme and chitosan degrading enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14051588A JP2685226B2 (en) | 1988-06-09 | 1988-06-09 | Method for separating chitinolytic enzyme and chitosan degrading enzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01309683A JPH01309683A (en) | 1989-12-14 |
| JP2685226B2 true JP2685226B2 (en) | 1997-12-03 |
Family
ID=15270449
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP14051588A Expired - Lifetime JP2685226B2 (en) | 1988-06-09 | 1988-06-09 | Method for separating chitinolytic enzyme and chitosan degrading enzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2685226B2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013077341A1 (en) * | 2011-11-21 | 2013-05-30 | 東レ株式会社 | Method for producing cellulase, and apparatus for said method |
-
1988
- 1988-06-09 JP JP14051588A patent/JP2685226B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01309683A (en) | 1989-12-14 |
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