JPH07177884A - Production of enzyme-containing composition - Google Patents
Production of enzyme-containing compositionInfo
- Publication number
- JPH07177884A JPH07177884A JP5346359A JP34635993A JPH07177884A JP H07177884 A JPH07177884 A JP H07177884A JP 5346359 A JP5346359 A JP 5346359A JP 34635993 A JP34635993 A JP 34635993A JP H07177884 A JPH07177884 A JP H07177884A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- water
- soluble polymer
- phase
- aqueous
- 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
Landscapes
- Detergent Compositions (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、酵素と水溶性高分子と
からなる酵素含有組成物の製造法に関する。本発明は特
に、洗剤と組合せて用いる酵素含有造粒物を製造する原
料として有利に利用できる酵素含有組成物の製造法に関
する。FIELD OF THE INVENTION The present invention relates to a method for producing an enzyme-containing composition comprising an enzyme and a water-soluble polymer. In particular, the present invention relates to a method for producing an enzyme-containing composition that can be advantageously used as a raw material for producing an enzyme-containing granule used in combination with a detergent.
【０００２】[0002]
【従来の技術】近年、衣料品洗浄用の粉末洗剤に、衣料
品に付いた汚れを分解して除去させることによって洗浄
作用を向上させるためにプロテアーゼ、セルラーゼなど
の酵素を配合することが一般的になっている。ただし、
酵素は洗剤の他の成分との接触により失活したり、また
取扱い者に刺激を与えることがあるため、通常はポリエ
チレングリコールなどのような水溶性高分子と共に造粒
を行ない、その造粒物として洗剤に配合されることが多
い。一方、通常の酵素は、酵素を生産する微生物（菌
体）を培養し、その培養液から濾過などの方法で菌体を
分離除去して得られる。2. Description of the Related Art In recent years, it has been common to mix powder detergents for washing clothes with enzymes such as protease and cellulase in order to improve the washing action by decomposing and removing stains on the clothes. It has become. However,
Enzymes may be inactivated by contact with other components of detergent and may cause irritation to the operator, so granulation is usually performed with water-soluble polymers such as polyethylene glycol. Often incorporated into detergents. On the other hand, a normal enzyme is obtained by culturing a microorganism (bacteria) that produces the enzyme, and separating and removing the cells from the culture solution by a method such as filtration.
【０００３】[0003]
【発明が解決しようとする課題】本発明は、酵素と水溶
性高分子とからなる酵素含有組成物の新規な製造法を提
供するものである。すなわち、従来より知られている菌
体を含む培養液から酵素を分離するための濾過操作は効
率が充分でなく、また生産された酵素が濾過基材に付着
するため、最終的に得られる酵素の収率が低下するとの
問題があった。従って、本発明は、菌体を含む培養液か
ら酵素を分離するための高効率な分離方法であって、か
つ生産された酵素を高い収率で回収することを可能にす
る方法を提供することを目的とする。また特に、本発明
は、洗剤と組合せて用いる酵素含有造粒物を製造する原
料として有利に利用できる酵素含有組成物を製造するた
めの方法を提供することも、その目的とする。The present invention provides a novel method for producing an enzyme-containing composition comprising an enzyme and a water-soluble polymer. That is, the filtration operation for separating the enzyme from the culture solution containing the conventionally known bacterial cells is not efficient, and the produced enzyme adheres to the filtration substrate, so that the enzyme finally obtained. However, there is a problem that the yield of the product decreases. Therefore, the present invention provides a highly efficient separation method for separating an enzyme from a culture solution containing bacterial cells, and a method that enables the produced enzyme to be recovered in a high yield. With the goal. It is also an object of the present invention, in particular, to provide a method for producing an enzyme-containing composition that can be advantageously used as a raw material for producing an enzyme-containing granule used in combination with a detergent.
【０００４】[0004]
【課題を解決するための手段】本発明は、菌体から産出
された酵素を、少なくとも一種類の水溶性高分子と、該
水溶性高分子と組み合わされて二相分配系を形成する他
の物質とから形成される水性二相分配系を利用して、酵
素を該水溶性高分子が分配される側の相に優先的に分配
し、該酵素を該水溶性高分子と共に分離し、乾燥するこ
とからなる酵素含有組成物の製造法にある。According to the present invention, an enzyme produced from a bacterial cell is combined with at least one water-soluble polymer to form a two-phase partition system. Utilizing an aqueous two-phase partition system formed from a substance, the enzyme is preferentially distributed to the phase on the side where the water-soluble polymer is distributed, the enzyme is separated together with the water-soluble polymer, and dried. The method for producing an enzyme-containing composition comprises:
【０００５】本発明で利用する水性二相分配系にはカチ
オン性界面活性剤を存在させることが好ましい。It is preferred that a cationic surfactant be present in the aqueous two-phase distribution system utilized in the present invention.
【０００６】本発明において使用される酵素の例として
は、衣類に付着した汚れを分解する作用を有する酵素
で、洗剤と併用することが知られているプロテアーゼ、
エステラーゼ、カルボヒドラーゼなどの酵素を挙げるこ
とができる。これらの酵素の具体例としては下記のもの
が挙げられる。 １）プロテアーゼ ペプシン、トリプシン、キモトリプシン、コラーゲナー
ゼ、ケラチナーゼ、エラスターゼ、スプリチシン、パパ
イン、アミノペプチターゼ、カルボキシペプチターゼ。 ２）エステラーゼ ガストリックリパーゼ、パンクレアチックリパーゼ、植
物リパーゼ類、ホスホリパーゼ類、コリンエステラーゼ
類、ホスホターゼ類 ３）カルボヒドラーゼ。 セルラーゼ、マルターゼ、サッカラーゼ、アミラーゼ、
ペクチナーゼ、α−及びβ−グリコシナーゼ。 なお、本発明で用いられる酵素は上記のものに限定され
るわけではなく、菌体から産出される酵素で、水溶性高
分子との造粒物として工業的な製品に用いられ得るもの
であれば、任意の酵素でよい。As an example of the enzyme used in the present invention, an enzyme having an action of decomposing dirt adhering to clothes and known to be used in combination with a detergent,
Enzymes such as esterase and carbohydrase can be mentioned. Specific examples of these enzymes include the following. 1) Proteases Pepsin, trypsin, chymotrypsin, collagenase, keratinase, elastase, spliticin, papain, aminopeptidase, carboxypeptidase. 2) Esterase gastric lipase, pancreatic lipase, plant lipase, phospholipase, cholinesterase, phosphotase 3) Carbohydrase. Cellulase, maltase, saccharase, amylase,
Pectinase, α- and β-glycosinase. It should be noted that the enzyme used in the present invention is not limited to the above-mentioned ones, and may be an enzyme produced from bacterial cells, which may be used in an industrial product as a granulated product with a water-soluble polymer. Any enzyme can be used.
【０００７】本発明で使用される水溶性高分子は、少な
くとも一種類の水溶性高分子と、該水溶性高分子と組み
合わされて二相分配系を形成する他の物質とを利用する
水性二相分離法の実施に際して用いられ得る水溶性高分
子である。この水性二層分離法（水性二相分配法あるい
は単に二相分配法ともいう）と水溶性高分子は、既に公
知である。The water-soluble polymer used in the present invention is an aqueous polymer that utilizes at least one water-soluble polymer and another substance that is combined with the water-soluble polymer to form a two-phase distribution system. It is a water-soluble polymer that can be used in carrying out the phase separation method. This aqueous two-layer separation method (also called an aqueous two-phase partition method or simply two-phase partition method) and a water-soluble polymer are already known.
【０００８】すなわち、二種類以上の物質の混合物から
目的の物質を分離する方法として、水性溶媒もしくは水
性溶液と有機溶媒とを用い、それらの溶媒への各物質の
分配係数の差を利用して分離（あるいは抽出）する方法
が一般的に利用されている。しかしながら、この有機溶
媒を用いる方法は、特に蛋白質などの生体高分子の分離
には使用できないことが多い。すなわち、多くの生体高
分子は有機溶媒との接触によって変性を起すためであ
る。従って、蛋白質などの生体高分子を、分配溶媒とし
て水のみを用い、水溶性高分子と、該水溶性高分子と組
み合されて二相分配系を形成する他の物質とを利用する
水性二相分離法が既に提案されており、実験室レベルで
は実際に使用されている。その分配系の例としては、二
種類の非電解性高分子の組合せ、非電解性高分子と電解
性高分子の組合せ、二種類の電解性高分子の組合せ、高
分子と低分子物質との組合せなどが知られており、分離
対象の目的物によって、各種使い分けられている。本発
明における水性二相分配系に用いる分配系の組合せの具
体例としては、ポリエチレングリコールとデキストラン
との組合せ、ポリエチレングリコールとリン酸塩（ナト
リウム塩、カリウム塩など）との組合せ、ポリエチレン
グリコールとクエン酸塩（ナトリウム塩、カリウム塩な
ど）との組合せ、ポリエチレングリコールと硫酸塩（ナ
トリウム塩、カリウム塩など）との組合せ、ポリエチレ
ングリコールとカルボキシメチルセルロースナトリウム
との組合せ、ポリエチレングリコールとデキストラン硫
酸塩との組合せ、ポリエチレングリコールとポリビニル
アルコールとの組合せなどを挙げることができ、これら
を目的に応じて使い分ける。また、塩類として、他のア
ルカリ金属塩、アルカリ土類金属塩、ハロゲン化物、チ
オシアン酸塩なども使用できる。なお、本発明で用いる
水溶性高分子はポリエチレングリコールであることが好
ましい。That is, as a method for separating a target substance from a mixture of two or more kinds of substances, an aqueous solvent or an aqueous solution and an organic solvent are used, and the difference in distribution coefficient of each substance to these solvents is utilized. A method of separating (or extracting) is generally used. However, this method using an organic solvent cannot often be used particularly for separating biopolymers such as proteins. That is, many biopolymers undergo denaturation upon contact with an organic solvent. Therefore, a water-based polymer that uses a biopolymer such as a protein only as water as a partitioning solvent and utilizes a water-soluble polymer and another substance that is combined with the water-soluble polymer to form a two-phase partition system. Phase separation methods have already been proposed and are in actual use at the laboratory level. Examples of the distribution system include a combination of two types of non-electrolytic polymers, a combination of non-electrolytic polymers and electrolytic polymers, a combination of two types of electrolytic polymers, a polymer and a low-molecular substance. Combinations and the like are known, and they are used in various ways depending on the object to be separated. Specific examples of the combination of partition systems used in the aqueous two-phase partition system of the present invention include a combination of polyethylene glycol and dextran, a combination of polyethylene glycol and a phosphate (sodium salt, potassium salt, etc.), a polyethylene glycol and a quenching salt. Combination with acid salt (sodium salt, potassium salt, etc.), combination with polyethylene glycol and sulfate salt (sodium salt, potassium salt, etc.), combination with polyethylene glycol and sodium carboxymethyl cellulose, combination with polyethylene glycol and dextran sulfate salt , A combination of polyethylene glycol and polyvinyl alcohol, etc., and these are used properly according to the purpose. Further, as the salts, other alkali metal salts, alkaline earth metal salts, halides, thiocyanates and the like can be used. The water-soluble polymer used in the present invention is preferably polyethylene glycol.
【０００９】すなわち、本発明によれば、水性二相分離
法を利用することにより、酵素を含む菌体の培養物から
直接、酵素を、その活性を低下させることなく、水溶性
高分子との混合物として分離し、これを公知の方法に従
って乾燥することによって、高収率で酵素含有組成物を
得ることができる。この酵素含有組成物はポリエチレン
グリコールなどの高分子物質との組成物の状態であるた
め、安全性や安定性が高く、またそのままの状態で、洗
剤に配合するための酵素造粒物の製造に、あるいは皮革
処理用の酵素製剤などの用途に利用できる。あるいは、
また酵素を含む菌体の培養物から菌体を公知の方法によ
って濾過除去したのち、濾液を水性二層分離法に供する
ことにより濾液中の着色物を、酵素が移行する水溶性高
分子相とは別の相に移行させることができ、従って、酵
素の精製が実現する。That is, according to the present invention, by utilizing the aqueous two-phase separation method, the enzyme can be directly treated with a water-soluble polymer from a culture of bacterial cells containing the enzyme without lowering its activity. The enzyme-containing composition can be obtained in high yield by separating it as a mixture and drying it according to a known method. Since this enzyme-containing composition is in a state of composition with a high molecular weight substance such as polyethylene glycol, it is highly safe and stable, and as it is, it can be used in the production of enzyme granules for incorporation into detergents. Alternatively, it can be used for applications such as enzyme preparations for leather treatment. Alternatively,
In addition, after the bacterial cells are removed by filtration from the culture of the bacterial cells containing the enzyme by a known method, the filtrate in the filtrate is subjected to an aqueous two-layer separation method to give a colored product in the filtrate, and a water-soluble polymer phase to which the enzyme migrates. Can be transferred to another phase, thus allowing purification of the enzyme.
【００１０】本発明の水性二相分離法の実施に際して
は、その二相分配系にカチオン界面活性剤を存在させる
ことが、水溶性高分子の側の相への酵素の優先的な移行
が容易となり、また他の側の相への着色成分の移行が促
進されるので特に好ましい。本発明において利用する二
相分配系に用いることのできるカチオン界面活性剤とし
ては以下のような分類のカチオン界面活性剤、そして具
体例を挙げることができる。ヘキサデシルトリメチルア
ンモニウム塩、テトラデシルトリメチルアンモニウム
塩、ドデシルトリメチルアンモニウム塩、ヘキサデシル
ピリジニウム塩などの第四級アミン塩、ドデシルアミン
塩、オクチルアミン塩、オクタデシルアミン塩などの第
一級アミン塩、その他各種の第二級アミン塩と第三級ア
ミン塩。本発明においてカチオン性界面活性剤は、水性
二相分配系を形成する成分（水を含む混合物）に対して
通常０．００１〜５重量％添加することが好ましい。In carrying out the aqueous two-phase separation method of the present invention, the presence of a cationic surfactant in the two-phase distribution system facilitates preferential transfer of the enzyme to the phase on the side of the water-soluble polymer. And is preferred because it facilitates the migration of the coloring component to the other phase. Examples of cationic surfactants that can be used in the two-phase distribution system used in the present invention include the following types of cationic surfactants and specific examples. Hexadecyltrimethylammonium salt, tetradecyltrimethylammonium salt, dodecyltrimethylammonium salt, hexadecylpyridinium salt and other quaternary amine salts, dodecylamine salt, octylamine salt, octadecylamine salt and other primary amine salts, and various other types. Secondary amine salts and tertiary amine salts of. In the present invention, the cationic surfactant is preferably added usually in an amount of 0.001 to 5% by weight based on the component (mixture containing water) forming the aqueous two-phase distribution system.
【００１１】なお、本発明の水性二相分離法では、一般
に、上層にポリエチレングリコールなどの水溶性高分子
と酵素とが優先的に集まって相を形成し、下層に、クエ
ン酸などの水性二相分配系の相手分の化合物と着色成分
が集まって相を形成する。なお、菌体の培養物をそのま
ま水性二相分離法に供した場合には、菌体は酵素と逆側
の層、すなわち下層に移行する。この時、カチオン性界
面活性剤が共存している場合には、それらの各成分の移
行、分離がより速やかに行なわれ、あるいはより優先的
に行なわれる。In the aqueous two-phase separation method of the present invention, generally, a water-soluble polymer such as polyethylene glycol and an enzyme are preferentially gathered in an upper layer to form a phase, and a lower layer is subjected to an aqueous two-phase separation such as citric acid. The compound of the counterpart of the phase distribution system and the coloring component gather to form a phase. When the culture of the bacterial cells is directly subjected to the aqueous two-phase separation method, the bacterial cells are transferred to the layer opposite to the enzyme, that is, the lower layer. At this time, when a cationic surfactant coexists, the transfer and separation of the respective components are carried out more quickly or preferentially.
【００１２】上記の水性二層分離法によって、酵素と水
溶性高分子とを一方の側（通常は上層）に移行させたの
ち、上層と下層とを分離し、その酵素と水溶性高分子と
を含む水相を一旦凍結乾燥などの方法で粉末化すること
によって、目的の酵素含有組成物を得ることができる。
上記の酵素含有組成物を洗剤と共に用いる酵素造粒物に
するには、たとえば、特開昭６２−２５７９９０公報に
記載の造粒操作を利用することができる。すなわち、前
記の酵素と水溶性高分子とを含む組成物（通常は粉末状
態にある）を、必要に応じて、親水性高分子を追加した
り、塩化カルシウムなどの酵素安定剤や硫酸ナトリウム
や塩化ナトリウムなどの粉末化助剤を添加したのち、こ
れを公知の造粒操作にかける。造粒方法の例としては、
撹拌転動などの造粒方法を挙げることができる。After transferring the enzyme and the water-soluble polymer to one side (usually the upper layer) by the above-mentioned aqueous two-layer separation method, the upper layer and the lower layer are separated, and the enzyme and the water-soluble polymer are separated. The target enzyme-containing composition can be obtained by once pulverizing the aqueous phase containing the by a method such as freeze-drying.
In order to make the above enzyme-containing composition into an enzyme granule for use with a detergent, for example, the granulation operation described in JP-A-62-257990 can be used. That is, if necessary, a hydrophilic polymer is added to a composition containing the enzyme and a water-soluble polymer (usually in a powder state), or an enzyme stabilizer such as calcium chloride or sodium sulfate or After adding a powdering aid such as sodium chloride, it is subjected to a known granulation operation. As an example of the granulation method,
A granulation method such as stirring and rolling can be mentioned.
【００１３】[0013]
［実施例１］細胞外プロテアーゼ産生バチルスエスピー
ＫＳＭ−Ｋ１６菌株（特開平４−３４９８８２号公報に
記載のもの）を、５リットル容量ジャーファメンタ中の
３リットルの培地（下記培地組成からなるもの）に接種
し、３０℃にて４８時間培養して、産出されたプロテア
ーゼを含む培養液を得た。この培養液を、精密濾過膜
（孔径：０．４５μｍ、アミコン株式会社製）を用いて
濾過して菌体を除去し、得られたプロテアーゼ溶液を凍
結乾燥して酵素粉末（濃褐色に着色したもの）を得た。[Example 1] An extracellular protease-producing Bacillus sp. KSM-K16 strain (described in JP-A-4-349882) was added to 3 liters of a medium (having the following medium composition) in a 5 liter volume jarfamentor. Was inoculated and cultured at 30 ° C. for 48 hours to obtain a culture solution containing the produced protease. This culture solution was filtered using a microfiltration membrane (pore size: 0.45 μm, manufactured by Amicon Co., Ltd.) to remove bacterial cells, and the obtained protease solution was freeze-dried to obtain an enzyme powder (colored dark brown). Stuff).
【００１４】 培地組成 グルコース ２．０ 重量％ ポリペプトンＳ １．０ 重量％ 酵母エキス（Ｄｉｆｃｏ） ０．０５重量％ リン酸一カリウム ０．１ 重量％ 硫酸マグネシウム・７Ｈ2 Ｏ ０．０２重量％ 炭酸ナトリウム １．０ 重量％ 上記の酵素粉末５０ｇを蒸留水１リットルに溶解し、下
記に記載するカゼイン法による酵素活性が２．０５Ｐ．
Ｕ．／ｍＬの酵素水溶液を調製した。 Medium composition Glucose 2.0% by weight Polypeptone S 1.0% by weight Yeast extract (Difco) 0.05% by weight Monopotassium phosphate 0.1% by weight Magnesium sulfate · 7H 2 O 0.02% by weight Sodium carbonate 1.0 wt% 50 g of the above enzyme powder was dissolved in 1 liter of distilled water, and the enzyme activity by the casein method described below was 2.05 p.
U. / ML of enzyme aqueous solution was prepared.
【００１５】プロテアーゼ酵素活性測定法（カゼイン
法） カゼイン基質による活性測定法：カゼインを１重量％含
む５０ｍＭほう酸−ＮａＯＨ緩衝液（ｐＨ１０．０）１
ｍＬを０．１ｍＬのプロテアーゼ含有溶液と混合し、４
０℃で１０分間反応させたのち、これに反応停止液
（０．１２３Ｍトリクロロ酢酸−０．２４６Ｍ酢酸ナト
リウム−０．３６９Ｍ酢酸）２ｍＬを加えて３０℃で２
０分間放置した。この放置した反応液を次に、濾紙（ワ
ットマン社製、Ｎｏ．２）を用いて濾過し、濾液中の蛋
白分解物をフォーリンローリー法の改良法によって測定
した。なお、１Ｐ．Ｕ．は、上記反応条件下において１
分間に１ミリモルのチロシンを遊離させる酵素量とし
た。 Protease enzyme activity assay (casein
Method) Method for measuring activity with casein substrate: 50 mM boric acid-NaOH buffer solution (pH 10.0) containing 1% by weight of casein 1
Mix mL with 0.1 mL of protease containing solution,
After reacting at 0 ° C for 10 minutes, 2 mL of a reaction stop solution (0.123M trichloroacetic acid-0.246M sodium acetate-0.369M acetic acid) was added to this, and the mixture was stirred at 30 ° C for 2 minutes.
It was left for 0 minutes. The reaction liquid left to stand was then filtered using a filter paper (Whatman, No. 2), and the protein degradation product in the filtrate was measured by an improved method of the Foreign Lowry method. In addition, 1P. U. Is 1 under the above reaction conditions.
It was defined as the amount of enzyme that liberates 1 mmol of tyrosine per minute.
【００１６】別に分液ロートを用意し、これに、上記の
酵素水溶液を入れたのち、水性二層分離の軽液側相形成
物質（ポリエチレングリコール（ＰＥＧ）６０００）
と、重液側相形成物質（リン酸二カリウム）とを、それ
ぞれ粉末状で５重量％、１５重量％（分液ロート内の酵
素水溶液の重量を基準とした）添加した。分液ロート内
の混合物を良く撹拌して、混合物内の粉末を溶解させた
のち、１５℃で２時間静置したところ、下層に着色成分
が優先的に移行して、下層の着色度が上層の着色度に比
べて明らかに高くなることが確認された。着色度が高い
下層のみを分液ロートから排出したところ、下層の液量
は８５０ｍＬであり、上層の液量は１５０ｍＬであり、
多量のポリエチレングリコールが含まれていた。また、
プロテアーゼ活性は、下層が０．３３Ｐ．Ｕ．であり、
上層が１１．６２Ｐ．Ｕ．であった。上層のプロテアー
ゼとポリエチレングリコールとを含む水溶液から凍結乾
燥法によってプロテアーゼとポリエチレングリコールと
を含む粉末状の酵素含有組成物を得た。上記の酵素含有
組成物粉末１００ｇに、塩化ナトリウム（平均粒子径６
１０μｍ、粒子径５００μｍ以下の粒子が１１重量％、
粒子径７００μｍ以上の粒子が９重量％のもの）５００
ｇと硫酸ナトリウム３５０ｇそして酸化チタン５０ｇを
添加し、ハイスピードミキサ（深江工業株式会社製、Ｆ
Ｓ−１０型）を利用して造粒して酵素含有造粒物を得
た。Separately, a separating funnel was prepared, and the above-mentioned aqueous enzyme solution was put therein, and then a light liquid side phase forming substance (polyethylene glycol (PEG) 6000) for aqueous two-layer separation was prepared.
5% by weight and 15% by weight (based on the weight of the enzyme aqueous solution in the separating funnel) of the heavy liquid side phase forming substance (dipotassium phosphate) were added in powder form. The mixture in the separatory funnel was well stirred to dissolve the powder in the mixture, and then the mixture was allowed to stand at 15 ° C for 2 hours, the coloring component was preferentially transferred to the lower layer, and the coloring degree of the lower layer was higher. It was confirmed that it was significantly higher than the coloring degree of. When only the lower layer having a high degree of coloring was discharged from the separating funnel, the lower layer had a liquid volume of 850 mL, and the upper layer had a liquid volume of 150 mL.
It contained a large amount of polyethylene glycol. Also,
The protease activity of the lower layer was 0.33P. U. And
The upper layer is 11.62P. U. Met. A powdery enzyme-containing composition containing a protease and polyethylene glycol was obtained from the upper layer aqueous solution containing a protease and polyethylene glycol by a freeze-drying method. To 100 g of the above enzyme-containing composition powder, sodium chloride (average particle size 6
11% by weight of particles having a particle size of 10 μm and a particle size of 500 μm or less,
9% by weight of particles having a particle size of 700 μm or more) 500
g, 350 g of sodium sulfate, and 50 g of titanium oxide were added, and a high speed mixer (Fukae Kogyo Co., Ltd., F
S-10 type) was used for granulation to obtain an enzyme-containing granulated product.
【００１７】［実施例２］細胞外セルラーゼ産生バチル
スエスピーＫＳＭ−６３５菌株（ＦＥＲＭ Ｐ−８８７
２）を、５リットル容量ジャーファメンタ中の２リット
ルの培地（下記培地組成からなるもの）に接種し、３０
℃にて７２時間培養して、産出されたセルラーゼを含む
培養液を得た。この培養液を、精密濾過膜（孔径：０．
４５μｍ、アミコン株式会社製）を用いて濾過して菌体
を除去し、得られた濃褐色のセルラーゼ溶液（下記のセ
ルラーゼ活性測定法による活性値：約３０００単位／
Ｌ）を得た。[Example 2] Extracellular cellulase-producing Bacillus sp. KSM-635 strain (FERM P-887)
2) was inoculated into 2 liters of a medium (consisting of the following medium composition) in a 5 liter volume jar-famentor, and 30
After culturing at 72 ° C. for 72 hours, a culture solution containing the produced cellulase was obtained. This culture solution was used as a microfiltration membrane (pore size: 0.
45 μm, manufactured by Amicon Co., Ltd.) to remove bacterial cells by filtration, and the dark brown cellulase solution obtained (activity value according to the following cellulase activity measuring method: about 3000 units /
L) was obtained.
【００１８】 培地組成 肉エキス １．５ 重量％ 酵母エキス ０．５ 重量％ カルボキシメチルセルロース １．０ 重量％ リン酸一カリウム ０．１ 重量％ 炭酸ナトリウム ０．７５重量％ Medium composition Meat extract 1.5% by weight Yeast extract 0.5% by weight Carboxymethyl cellulose 1.0% by weight Monopotassium phosphate 0.1% by weight Sodium carbonate 0.75% by weight
【００１９】セルラーゼ酵素活性測定法（ＣＭＣ活性測
定法） ２．５％カルボキシメチルセルロース（ＣＭＣ）０．２
ｍＬ、０．５Ｍグリシン緩衝液（ｐＨ９．０）０．１ｍ
Ｌ、および脱イオン水０．１ｍＬからなる基質溶液に酵
素溶液０．１ｍＬを加え、４０℃で２０分間反応させ
る。反応後、ＤＮＳ（３，５−ジニトロサリチル酸）法
を利用して還元糖の定量を行なった。すなわち、反応液
０．５ｍＬに対してＤＮＳ試薬１ｍＬを加え５分間、１
００℃で加熱して、発色させる。次いで冷却したのち、
４．５ｍＬの脱イオン水を加えて希釈する。この条件で
１分間でグルコール換算で１μモルの還元糖を遊離させ
る酵素量を１単位とする。 Cellulase enzyme activity assay (CMC activity assay
Standard method) 2.5% carboxymethyl cellulose (CMC) 0.2
mL, 0.5 M glycine buffer (pH 9.0) 0.1 m
0.1 mL of the enzyme solution is added to the substrate solution consisting of L and 0.1 mL of deionized water, and the mixture is reacted at 40 ° C. for 20 minutes. After the reaction, the reducing sugar was quantified using the DNS (3,5-dinitrosalicylic acid) method. That is, 1 mL of the DNS reagent was added to 0.5 mL of the reaction solution for 1 minute for 1 minute.
Heat at 00 ° C. to develop color. Then, after cooling,
Dilute by adding 4.5 mL deionized water. Under this condition, the amount of the enzyme that liberates 1 μmol of reducing sugar in glucose conversion per minute is 1 unit.
【００２０】別に分液ロートを用意し、これに前記のセ
ルラーゼ溶液を入れたのち、水性二層分離の軽液側相形
成物質（ポリエチレングリコール（ＰＥＧ）６０００）
と、重液側相形成物質（デキストラン、分子量１０万〜
２０万）とを、それぞれ粉末状で４重量％、１４重量％
（分液ロート内のセルラーゼ溶液の重量を基準）添加し
た。さらに、セルラーゼの上層移行促進剤としてリン酸
二カリウムを８重量％となるように粉末状で添加したの
ち、分液ロート内の混合物を良く撹拌して、混合物内の
粉末を溶解させたのち、１５℃で２時間静置したとこ
ろ、下層に着色成分が優先的に移行して、下層の着色度
が上層の着色度に比べて明らかに高くなることが確認さ
れた。着色度が高い下層のみを分液ロートから排出した
ところ、下層の液量は１８００ｍＬであり、上層の液量
は２００ｍＬであり、多量のポリエチレングリコールが
含まれていた。また、セルラーゼ活性は、下層が７７０
単位／Ｌであり、上層が２３０００単位／Ｌであった。
上層のセルラーゼとポリエチレングリコールとを含む水
溶液から凍結乾燥法によってセルラーゼとポリエチレン
グリコールとを含む粉末状の酵素含有組成物を得た。上
記の酵素含有組成物粉末１００ｇに、塩化ナトリウム
（平均粒子径６１０μｍ、粒子径５００μｍ以下の粒子
が１１重量％、粒子径７００μｍ以上の粒子が９重量％
のもの）５００ｇと硫酸ナトリウム３５０ｇそして酸化
チタン５０ｇを添加し、ハイスピードミキサを利用して
造粒し、酵素含有造粒物を得た。Separately, a separating funnel was prepared, and the above cellulase solution was put into the separating funnel. Then, a light liquid side phase forming substance (polyethylene glycol (PEG) 6000) for aqueous two-layer separation was prepared.
And heavy liquid side phase forming substance (dextran, molecular weight 100,000 ~
200,000) and 4% by weight and 14% by weight in powder form, respectively.
(Based on the weight of the cellulase solution in the separating funnel) was added. Furthermore, after adding dipotassium phosphate as a cellulase upper layer migration promoter in a powder form so as to be 8% by weight, after thoroughly stirring the mixture in the separating funnel to dissolve the powder in the mixture, When left at 15 ° C. for 2 hours, it was confirmed that the coloring components were preferentially transferred to the lower layer, and the coloring degree of the lower layer was obviously higher than that of the upper layer. When only the lower layer having a high degree of coloring was discharged from the separating funnel, the lower layer had a liquid volume of 1800 mL, the upper layer had a liquid volume of 200 mL, and contained a large amount of polyethylene glycol. In addition, the cellulase activity is 770 in the lower layer.
Unit / L, and the upper layer was 23000 unit / L.
A powdery enzyme-containing composition containing cellulase and polyethylene glycol was obtained from the upper layer aqueous solution containing cellulase and polyethylene glycol by freeze-drying. Sodium chloride (11% by weight of particles having an average particle size of 610 μm and 500 μm or less and 9% by weight of particles having a particle size of 700 μm or more) was added to 100 g of the above enzyme-containing composition powder.
500 g), sodium sulfate (350 g) and titanium oxide (50 g) were added and granulated using a high speed mixer to obtain an enzyme-containing granulated product.
【００２１】［実施例３］細胞外プロテアーゼ産生バチ
ルスエスピーＫＳＭ−Ｋ１６菌株（特開平４−３４９８
８２号公報に記載のもの）を、５００ｍＬ容量＝坂口フ
ラスコ中の液体培地（実施例１に記載の培地組成からな
るもの）５０ｍＬに接種し、３０℃にて４８時間培養し
て、培養液を得た。別に、分液ロートを用意し、これ
に、水性二層分離の軽液側相形成物質（ポリエチレング
リコール（ＰＥＧ）４０００）と、重液側相形成物質
（クエン酸ナトリウム二水和物）とを、それぞれ粉末状
で１４重量％入れた。次いで、カチオン性界面活性剤
（ヘキサデシルトリメチルアンモニウムブロミド、ＨＤ
ＡＢ）を、０．５重量％添加した（無添加のものは比較
対照用）。この分液ロートに前記の培養液を添加し、撹
拌混合して試験管内の粉末を溶解させて、５００ｍＬの
混合液とした。この分液ロートを１５℃で１時間静置し
て菌体を下層に分離した後、上層のＰＥＧ−プロテアー
ゼ溶液を分離した。取り出された上層液量は２５０ｍＬ
であり、下層液量は２５０ｍＬであった。また、取り出
された上層液には、ＰＥＧ４０００が約３５重量％、ク
エン酸塩が５重量％、そしてＨＤＡＢが０．１重量％含
まれており、その着色度は下層液に比べて明らかに低下
していた。得られた上層のプロテアーゼとポリエチレン
グリコールとを含む水溶液を凍結乾燥して粉末状の酵素
含有組成物を得た。上記の酵素含有組成物に、塩化ナト
リウム（平均粒子径６１０μｍ、粒子径５００μｍ以下
の粒子が１１重量％、粒子径７００μｍ以上の粒子が９
重量％のもの）と硫酸ナトリウムそして酸化チタンを添
加し、ハイスピードミキサを利用して造粒して酵素含有
造粒物を得た。[Example 3] Extracellular protease-producing Bacillus sp. KSM-K16 strain (JP-A-4-3498)
No. 82) is inoculated into 50 mL of a liquid medium (consisting of the medium composition described in Example 1) in a 500 mL capacity = Sakaguchi flask and cultured at 30 ° C. for 48 hours to prepare a culture solution. Obtained. Separately, a separating funnel was prepared, and a light liquid side phase forming substance (polyethylene glycol (PEG) 4000) for aqueous two-layer separation and a heavy liquid side phase forming substance (sodium citrate dihydrate) were added to the funnel. , 14 wt% in powder form, respectively. Then, a cationic surfactant (hexadecyltrimethylammonium bromide, HD
AB) was added in an amount of 0.5% by weight (the one without addition was used as a comparative control). The above culture solution was added to this separating funnel, and the mixture was stirred and mixed to dissolve the powder in the test tube to prepare a 500 mL mixed solution. The separating funnel was allowed to stand at 15 ° C. for 1 hour to separate the bacterial cells into the lower layer, and then the upper layer PEG-protease solution was separated. The amount of the upper layer liquid taken out is 250 mL
And the lower layer liquid volume was 250 mL. In addition, the extracted upper layer liquid contained about 35% by weight of PEG4000, 5% by weight of citrate, and 0.1% by weight of HDAB, and the degree of coloring was clearly lower than that of the lower layer liquid. Was. The obtained upper layer aqueous solution containing protease and polyethylene glycol was freeze-dried to obtain a powdery enzyme-containing composition. Sodium chloride (average particle size 610 μm, particles having a particle size of 500 μm or less was 11% by weight, and particles having a particle size of 700 μm or more were 9% in the above enzyme-containing composition.
(Wt%), sodium sulfate and titanium oxide were added, and granulated using a high speed mixer to obtain an enzyme-containing granulated product.
【００２２】［実施例４］バチルス属の菌株を培養した
のち、限外濾過膜を用いて培養液の菌体濁度を約５０に
調整した。このようにして調製した菌体濁度調整培養液
に、ポリエチレングリコール（ＰＥＧ）４０００とリン
酸ナトリウムとをそれぞれ１４重量％となるように添加
し、次いでヘキサデシルトリメチルアンモニウムブロミ
ドを０．５重量％の濃度になるように添加した。次い
で、試料液を２５℃で５時間静置し、分離した上層と下
層のそれぞれの菌体濁度を測定したところ、上層の濁度
はほぼ０であり、菌体の大部分が下層に移行したことが
確認された。また上層の着色度は下層液に比べて明らか
に低下していた。得られた上層のプロテアーゼとポリエ
チレングリコールとを含む水溶液から凍結乾燥法によっ
てプロテアーゼとポリエチレングリコールとを含む粉末
状の酵素含有組成物を得た。上記の酵素含有組成物粉末
に、塩化ナトリウム（平均粒子径６１０μｍ、粒子径５
００μｍ以下の粒子が１１重量％、粒子径７００μｍ以
上の粒子が９重量％のもの）と硫酸ナトリウムそして酸
化チタンを添加し、ハイスピードミキサを利用して造粒
して、酵素含有造粒物を得た。[Example 4] After culturing a Bacillus strain, the turbidity of the culture was adjusted to about 50 using an ultrafiltration membrane. Polyethylene glycol (PEG) 4000 and sodium phosphate were added to the thus-prepared cell turbidity-adjusting culture solution so that each of them would be 14% by weight, and then hexadecyltrimethylammonium bromide was added at 0.5% by weight. Was added so as to have a concentration of. Then, the sample solution was allowed to stand at 25 ° C for 5 hours, and the turbidity of the upper and lower separated cells was measured. The turbidity of the upper layer was almost 0, and most of the cells migrated to the lower layer. It was confirmed that it did. In addition, the degree of coloring of the upper layer was obviously lower than that of the lower layer liquid. A powdery enzyme-containing composition containing a protease and polyethylene glycol was obtained from the resulting aqueous solution containing the protease and polyethylene glycol by a freeze-drying method. Sodium chloride (average particle size 610 μm, particle size 5
11% by weight of particles of 00 μm or less and 9% by weight of particles of 700 μm or more in diameter), sodium sulfate and titanium oxide are added and granulated using a high speed mixer to obtain an enzyme-containing granulated product. Obtained.
【００２３】[0023]
【発明の効果】本発明の水性二相分離方法を利用した酵
素含有組成物の製造法を利用することにより、菌体から
分離した酵素が水溶性高分子との組成物として高収率で
回収することができる。この酵素含有組成物は、洗剤に
配合するのに適した造粒物の製造に特に適している。ま
た、本発明により得られる酵素含有組成物は、それ自体
で水溶性高分子物質を含有しているため、皮革処理用の
酵素製剤などとしても有用である。Industrial Applicability By utilizing the method for producing an enzyme-containing composition using the aqueous two-phase separation method of the present invention, the enzyme separated from the bacterial cells is recovered in a high yield as a composition with a water-soluble polymer. can do. This enzyme-containing composition is particularly suitable for the production of granulates suitable for incorporation into detergents. Further, the enzyme-containing composition obtained by the present invention contains a water-soluble polymer substance by itself, and is therefore also useful as an enzyme preparation for leather treatment.
Claims (2)
一種類の水溶性高分子と、該水溶性高分子と組み合わさ
れて二相分配系を形成する他の物質とから形成される水
性二相分配系を利用して、酵素を該水溶性高分子が分配
される側の相に優先的に分配し、該酵素を該水溶性高分
子と共に分離し、乾燥することからなる酵素含有組成物
の製造法。1. An aqueous solution comprising an enzyme produced from bacterial cells, which is formed from at least one water-soluble polymer and another substance which is combined with the water-soluble polymer to form a two-phase partition system. An enzyme-containing composition comprising: preferentially partitioning an enzyme into a phase on the side where the water-soluble polymer is distributed using a phase partitioning system, separating the enzyme together with the water-soluble polymer, and drying. Manufacturing method.
剤を存在させることを特徴とする請求項１に記載の酵素
含有組成物の製造法。2. The method for producing an enzyme-containing composition according to claim 1, wherein a cationic surfactant is present in the aqueous two-phase partition system.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP34635993A JP3152826B2 (en) | 1993-12-22 | 1993-12-22 | Method for producing enzyme-containing composition |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP34635993A JP3152826B2 (en) | 1993-12-22 | 1993-12-22 | Method for producing enzyme-containing composition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH07177884A true JPH07177884A (en) | 1995-07-18 |
| JP3152826B2 JP3152826B2 (en) | 2001-04-03 |
Family
ID=18382883
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP34635993A Expired - Fee Related JP3152826B2 (en) | 1993-12-22 | 1993-12-22 | Method for producing enzyme-containing composition |
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| Country | Link |
|---|---|
| JP (1) | JP3152826B2 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999001531A1 (en) * | 1997-07-02 | 1999-01-14 | The Procter & Gamble Company | Dishwashing compositions comprising a phospholipase and an amylase |
| WO2008040466A1 (en) | 2006-10-02 | 2008-04-10 | Ab Enzymes Gmbh | Clonation, expression and use of acid phospholipases |
| EP2113563A2 (en) | 1998-11-27 | 2009-11-04 | Novozymes A/S | Lipolytic enzyme variants |
| US7998715B2 (en) | 2005-09-20 | 2011-08-16 | Asahi Breweries, Ltd. | Method of producing liquid koji having enhanced plant fiber degeneration enzyme, liquid koji obtained by the method and use thereof |
| US8124374B2 (en) | 2005-10-12 | 2012-02-28 | Asahi Breweries, Ltd. | Method of producing recombinant protein |
| US8715979B2 (en) | 2005-10-05 | 2014-05-06 | Asahi Breweries, Ltd. | Method of producing filamentous fungus culture product |
| US8802170B2 (en) | 2004-04-09 | 2014-08-12 | Asahi Breweries, Ltd. | Method of manufacturing liquid koji |
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1993
- 1993-12-22 JP JP34635993A patent/JP3152826B2/en not_active Expired - Fee Related
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999001531A1 (en) * | 1997-07-02 | 1999-01-14 | The Procter & Gamble Company | Dishwashing compositions comprising a phospholipase and an amylase |
| EP2298873A1 (en) | 1998-11-27 | 2011-03-23 | Novozymes A/S | Lipolytic enzyme variants |
| EP2287298A1 (en) | 1998-11-27 | 2011-02-23 | Novozymes A/S | Lipolytic enzyme variants |
| EP2302043A2 (en) | 1998-11-27 | 2011-03-30 | Novozymes A/S | Lipolytic enzyme variants |
| EP2287297A1 (en) | 1998-11-27 | 2011-02-23 | Novozymes A/S | Lipolytic enzyme variants |
| EP2302044A1 (en) | 1998-11-27 | 2011-03-30 | Novozymes A/S | Lipolytic enzyme variants |
| EP2290059A1 (en) | 1998-11-27 | 2011-03-02 | Novozymes A/S | Lipolytic enzyme variants |
| EP2290058A1 (en) | 1998-11-27 | 2011-03-02 | Novozymes A/S | Lipolytic enzyme variants |
| EP2716753A1 (en) | 1998-11-27 | 2014-04-09 | Novozymes A/S | Lipolytic enzyme variants |
| EP2236602A1 (en) | 1998-11-27 | 2010-10-06 | Novozymes A/S | Lipolytic enzyme variants |
| EP2113563A2 (en) | 1998-11-27 | 2009-11-04 | Novozymes A/S | Lipolytic enzyme variants |
| US8802170B2 (en) | 2004-04-09 | 2014-08-12 | Asahi Breweries, Ltd. | Method of manufacturing liquid koji |
| US7998715B2 (en) | 2005-09-20 | 2011-08-16 | Asahi Breweries, Ltd. | Method of producing liquid koji having enhanced plant fiber degeneration enzyme, liquid koji obtained by the method and use thereof |
| US8715979B2 (en) | 2005-10-05 | 2014-05-06 | Asahi Breweries, Ltd. | Method of producing filamentous fungus culture product |
| US8124374B2 (en) | 2005-10-12 | 2012-02-28 | Asahi Breweries, Ltd. | Method of producing recombinant protein |
| WO2008040466A1 (en) | 2006-10-02 | 2008-04-10 | Ab Enzymes Gmbh | Clonation, expression and use of acid phospholipases |
| US8653241B2 (en) | 2006-10-02 | 2014-02-18 | Ab Enzymes Gmbh | Phospholipase polypeptide and a DNA encoding same |
| US7993876B2 (en) | 2006-10-02 | 2011-08-09 | Ab Enzymes Gmbh | DNA encoding phospholipases and methods of using same |
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| JP3152826B2 (en) | 2001-04-03 |
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