JPH03164170A - Purification of enzyme - Google Patents
Purification of enzymeInfo
- Publication number
- JPH03164170A JPH03164170A JP1301969A JP30196989A JPH03164170A JP H03164170 A JPH03164170 A JP H03164170A JP 1301969 A JP1301969 A JP 1301969A JP 30196989 A JP30196989 A JP 30196989A JP H03164170 A JPH03164170 A JP H03164170A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- membrane
- cells
- enzymes
- hydroxyl group
- 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
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 20
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 20
- 238000000746 purification Methods 0.000 title claims description 5
- 239000012528 membrane Substances 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 18
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 7
- 230000007935 neutral effect Effects 0.000 claims abstract description 7
- 238000000855 fermentation Methods 0.000 claims description 10
- 230000004151 fermentation Effects 0.000 claims description 10
- 239000012982 microporous membrane Substances 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 239000011148 porous material Substances 0.000 claims description 6
- 229920001600 hydrophobic polymer Polymers 0.000 claims description 3
- 230000008569 process Effects 0.000 abstract description 3
- 229920001477 hydrophilic polymer Polymers 0.000 abstract 1
- 239000000758 substrate Substances 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- -1 but in recent years Proteins 0.000 description 6
- 108010073038 Penicillin Amidase Proteins 0.000 description 3
- 239000004698 Polyethylene Substances 0.000 description 3
- 230000035699 permeability Effects 0.000 description 3
- 229920000573 polyethylene Polymers 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 150000001336 alkenes Chemical class 0.000 description 2
- 238000011001 backwashing Methods 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 239000012510 hollow fiber Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 238000002834 transmittance Methods 0.000 description 2
- LIKMAJRDDDTEIG-UHFFFAOYSA-N 1-hexene Chemical compound CCCCC=C LIKMAJRDDDTEIG-UHFFFAOYSA-N 0.000 description 1
- 241000194107 Bacillus megaterium Species 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- UUAGAQFQZIEFAH-UHFFFAOYSA-N chlorotrifluoroethylene Chemical group FC(F)=C(F)Cl UUAGAQFQZIEFAH-UHFFFAOYSA-N 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000003495 polar organic solvent Substances 0.000 description 1
- 229920002492 poly(sulfone) Polymers 0.000 description 1
- 229920000098 polyolefin Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- BFKJFAAPBSQJPD-UHFFFAOYSA-N tetrafluoroethene Chemical group FC(F)=C(F)F BFKJFAAPBSQJPD-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野］
本発明は発酵液から連続的に酵素を精製する方法に関す
るものである．さらに詳しくは、発酵液中から、菌体と
酵素とを分離する方法に関するものである。[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for continuously purifying enzymes from fermentation broth. More specifically, the present invention relates to a method for separating bacterial cells and enzymes from a fermentation liquid.
酵素の工業的精製法としては、従来、極性有機溶剤によ
る沈澱法や硫酸アンモニウムなどによる塩析法が知られ
ているが、近年、複雑な工程を必要としない限外炉過膜
による酵素の精製方法が注目されている。しかしながら
、これまでの限外炉過膜は、酵素の膜への吸着、耐熱性
の点で必ずしも充分でなく、さらに合理化された精製方
法の出現が期待されている。Conventionally, precipitation methods using polar organic solvents and salting-out methods using ammonium sulfate have been known as industrial purification methods for enzymes, but in recent years, enzyme purification methods using ultrafilter membranes that do not require complicated processes have been developed. is attracting attention. However, conventional ultrafilter membranes are not necessarily sufficient in terms of adsorption of enzymes to the membrane and heat resistance, and the emergence of more streamlined purification methods is expected.
本発明はシンプルな工程で、かつ簡便な手法で、発酵液
中から菌体と酵素とを分離する方法を提供するものであ
る。The present invention provides a method for separating bacterial cells and enzymes from a fermentation liquid using simple steps and a simple method.
本発明は、菌体と酵素を含む発酵液を、疎水性ポリマー
からなる基材膜に中性ヒドロキシル基を有するグラフト
鎖が結合された、平均孔径０． Ｏｆ〜１．０μｍの微
多孔膜に透過させることを特徴とする酵素の精製方法で
ある。In the present invention, a fermentation liquid containing bacterial cells and enzymes is transferred to a base membrane made of a hydrophobic polymer with an average pore diameter of 0. This is a method for purifying an enzyme, which is characterized by permeating it through a microporous membrane of ~1.0 μm.
本発明に適用される発酵液は、菌体と酵素を含むもので
あり、酵素は典型的には菌体外酵素で、分子量がｌＯ万
を越えないものが好ましい。The fermentation liquid applied to the present invention contains microbial cells and enzymes, and the enzyme is typically an extracellular enzyme, preferably having a molecular weight not exceeding 10,000.
発酵液は微多孔膜に透過させて、菌体と酵素とを分離す
る．発酵液は水を主体とするので、膜は親水性であるこ
とが必要である。膜の素材が親水性であることは必要で
ない。膜の強度、耐薬品性などの点から、疎水性ポリマ
ーからなる基材膜の少なくとも表面に、親水性を付与し
た膜のほうがむしろ適当である。The fermentation liquid is passed through a microporous membrane to separate the bacterial cells and enzymes. Since the fermentation liquid consists mainly of water, the membrane needs to be hydrophilic. It is not necessary that the membrane material be hydrophilic. From the viewpoint of film strength, chemical resistance, etc., a film in which at least the surface of a base film made of a hydrophobic polymer is imparted with hydrophilic properties is more suitable.
基材膜の素材としては、ポリエチレンやポリプロピレン
などのポリオレフイン；エチレン、プロピレン、ブテン
、ベンテン、ヘキセンなどのオレフィンから選ばれる１
種とテトラフルオ口エチレン、クロロトリフルオロエチ
レンなどのハロゲン化オレフィンとの共重合体；ポリフ
ツ化ビニリデン；またはポリスルホンがあげられるが、
ポリエチレンが最も好ましい。The material for the base film is selected from polyolefins such as polyethylene and polypropylene; olefins such as ethylene, propylene, butene, bentene, and hexene.
Examples include copolymers of seeds and halogenated olefins such as tetrafluoroethylene and chlorotrifluoroethylene; polyvinylidene fluoride; and polysulfone.
Polyethylene is most preferred.
基材膜に親水性を付与する手段としては、基材膜に中性
ヒドロキシル基をもつグラフト鎖を結合させる方法があ
げられる。中性ヒドロキシル基をもつグラフト鎖として
はポリビニルアルコールが好ましい。As a means for imparting hydrophilicity to the base film, there may be mentioned a method of bonding a graft chain having a neutral hydroxyl group to the base film. Polyvinyl alcohol is preferred as the graft chain having neutral hydroxyl groups.
中性ヒドロキシル基の量は、親水性微多孔膜１ｇ当り０
．１〜２０ミリ当量、好ましくは１〜１０ミリ当量の範
囲から選ばれる。The amount of neutral hydroxyl groups is 0 per gram of hydrophilic microporous membrane.
．． It is selected from the range of 1 to 20 milliequivalents, preferably 1 to 10 milliequivalents.
親水性微多孔膜は中空糸状であることが好ましい。内径
０．１＝ １ ０ １Ｉｎ，膜厚０．０５〜５ｍの形状
を有するものが効率上好ましい．
親水性微多孔膜は、平均孔径が０．０１〜１μｍの範囲
にあることが好ましい。ここで平均孔径とは、ＡＳＴＭ
Ｆ ３１６−７０に記載されている方法による値を示し
ており、通常エアーフロー法と呼ばれ、空気圧を変えて
乾燥膜と湿潤膜の空気透過流束を測定し、その比から求
めるものである。また、多孔膜の空孔率は２０〜９０％
、好ましくは５０〜９０％の範囲から選ばれる。空孔率
は、あらかじめ多孔膜を水等の液体に浸漬し、その後乾
燥させてその前後の重量変化から測定したものである。The hydrophilic microporous membrane is preferably in the form of hollow fibers. In terms of efficiency, it is preferable to have a shape with an inner diameter of 0.1=101 In and a film thickness of 0.05 to 5 m. The hydrophilic microporous membrane preferably has an average pore diameter in the range of 0.01 to 1 μm. Here, the average pore size is defined as ASTM
The values are shown based on the method described in F 316-70, which is usually called the air flow method, and is calculated by measuring the air permeation flux through the dry membrane and wet membrane by changing the air pressure and calculating the ratio. . In addition, the porosity of the porous membrane is 20 to 90%.
, preferably from the range of 50 to 90%. The porosity is measured by immersing the porous membrane in a liquid such as water in advance, then drying it, and measuring the weight change before and after that.
本発明で使用される微多孔膜は、乾燥状態において膜の
片側から水圧をかけ、膜のもう一方の面へ透水が認めら
れるいわゆる透水圧が０．８ ｋｇ／ｃｆｆｌ以下であ
り、また、乾燥膜における２５’Ｃ，１ｋｇ／ｄでの純
水の透水率を、あらかじめエタノールで処理した膜の透
水率と比較した値（ａ水化率）は、１５％以上である。The microporous membrane used in the present invention has a so-called water permeability pressure of 0.8 kg/cffl or less, in which water permeates to the other side of the membrane when water pressure is applied from one side of the membrane in a dry state. The water permeability of pure water in the membrane at 25'C and 1 kg/d is compared with the water permeability of a membrane treated with ethanol in advance (water conversion rate a), which is 15% or more.
本発明によれば、微多孔膜への酵素の吸着が抑制され、
高いＦｌｕｘが維持できる。According to the present invention, adsorption of enzymes to the microporous membrane is suppressed,
High flux can be maintained.
実施例
ポリエチレンからなる中空糸状多孔性基材膜に、ポリビ
ニルアルコールが中性ヒドロキシル基量として膜１ｇ当
り４．８ξり当量結合した親水性微多孔膜（平均孔径０
．２５μｍ、空孔率７０％）を内蔵した膜面積０．０９
ｒｒｆのモジュールを用いて、ペニシリンアシラーゼ
を産生ずる菌体（バチルスメガテリウム）の発酵液を炉
過した。Example Hydrophilic microporous membrane (average pore size 0
．． 25μm, porosity 70%) membrane area 0.09
Using the rrf module, a fermentation solution of a bacterial cell (Bacillus megaterium) producing penicillin acylase was filtered.
炉過圧Ｐｉ／Ｐｏ＝０．６／０．４ ｋｇ／ｃｔＡ、炉
過６分毎に水による逆洗を５秒間（逆洗圧０．７ｋｇ／
ｃｉｉ）行いつつ、５４分間が過を行った。Furnace overpressure Pi/Po=0.6/0.4 kg/ctA, backwashing with water for 5 seconds every 6 minutes (backwashing pressure 0.7kg/ctA)
cii) Fifty-four minutes passed during the process.
平均炉過流量は７８１／ｎｆ・ｈｒ，濃縮倍率は３倍で
、その透過率は６１％であった。得られたペニシリンア
シラーゼの分子量は９万であった．比較例
中性ヒドロキシル基が結合されていないポリエチレン中
空糸状微多孔膜（平均孔径０．２５μｍ、空孔率７０％
）を用いた以外は、実施例と同一の条件で実施例と同一
の発酵液を７０分間が過した。The average furnace flow rate was 781/nf·hr, the concentration ratio was 3 times, and the transmittance was 61%. The molecular weight of the obtained penicillin acylase was 90,000. Comparative Example Polyethylene hollow fiber microporous membrane to which neutral hydroxyl groups are not bonded (average pore diameter 0.25 μm, porosity 70%)
) was used for 70 minutes using the same fermentation liquid as in the example under the same conditions as in the example.
平均炉過流量は４５ １／ｒｒｒ −ｈｒ， ＠縮倍率
は２．４倍で、その透過率は４２％であった。得られた
ペニシリンアシラーゼの分子量は９万であった。The average furnace flow rate was 45 1/rrr -hr, the reduction ratio was 2.4 times, and the transmittance was 42%. The molecular weight of the obtained penicillin acylase was 90,000.
Claims (1)
材膜に中性ヒドロキシル基を有するグラフト鎖が結合さ
れた、平均孔径０．０１〜１．０μｍの微多孔膜に透過
させることを特徴とする酵素の精製方法The fermentation liquid containing bacterial cells and enzymes is permeated through a microporous membrane with an average pore diameter of 0.01 to 1.0 μm, which has a graft chain having a neutral hydroxyl group bonded to a base membrane made of a hydrophobic polymer. Featured enzyme purification method
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1301969A JP2886579B2 (en) | 1989-11-22 | 1989-11-22 | Enzyme purification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1301969A JP2886579B2 (en) | 1989-11-22 | 1989-11-22 | Enzyme purification method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH03164170A true JPH03164170A (en) | 1991-07-16 |
| JP2886579B2 JP2886579B2 (en) | 1999-04-26 |
Family
ID=17903311
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1301969A Expired - Fee Related JP2886579B2 (en) | 1989-11-22 | 1989-11-22 | Enzyme purification method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2886579B2 (en) |
-
1989
- 1989-11-22 JP JP1301969A patent/JP2886579B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2886579B2 (en) | 1999-04-26 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| LAPS | Cancellation because of no payment of annual fees |