JPH1036676A - Method for concentrating aqueous protein solution - Google Patents
Method for concentrating aqueous protein solutionInfo
- Publication number
- JPH1036676A JPH1036676A JP21522496A JP21522496A JPH1036676A JP H1036676 A JPH1036676 A JP H1036676A JP 21522496 A JP21522496 A JP 21522496A JP 21522496 A JP21522496 A JP 21522496A JP H1036676 A JPH1036676 A JP H1036676A
- Authority
- JP
- Japan
- Prior art keywords
- concentration
- solution
- aqueous
- protein
- aqueous solution
- 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
- 239000012460 protein solution Substances 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 title claims description 26
- 239000007864 aqueous solution Substances 0.000 claims abstract description 29
- 238000000502 dialysis Methods 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 16
- 239000012528 membrane Substances 0.000 claims abstract description 11
- 229920001223 polyethylene glycol Polymers 0.000 claims description 28
- 108010022355 Fibroins Proteins 0.000 claims description 20
- 229920000642 polymer Polymers 0.000 claims description 13
- 239000002202 Polyethylene glycol Substances 0.000 claims description 11
- 229920000570 polyether Polymers 0.000 claims description 8
- 229920005862 polyol Polymers 0.000 claims description 6
- 150000003077 polyols Chemical class 0.000 claims description 6
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 abstract description 46
- 239000000243 solution Substances 0.000 abstract description 11
- 229920005601 base polymer Polymers 0.000 abstract 4
- 239000012141 concentrate Substances 0.000 abstract 1
- 229920001577 copolymer Polymers 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 238000003756 stirring Methods 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 230000007423 decrease Effects 0.000 description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N monopropylene glycol Natural products CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- 239000001913 cellulose Substances 0.000 description 5
- 229920002678 cellulose Polymers 0.000 description 5
- 238000007654 immersion Methods 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 238000001556 precipitation Methods 0.000 description 5
- 229920005604 random copolymer Polymers 0.000 description 5
- 238000001879 gelation Methods 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 238000012937 correction Methods 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical group C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- HQPMKSGTIOYHJT-UHFFFAOYSA-N ethane-1,2-diol;propane-1,2-diol Chemical compound OCCO.CC(O)CO HQPMKSGTIOYHJT-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 150000002334 glycols Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000005185 salting out Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- AWFYPPSBLUWMFQ-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(1,4,6,7-tetrahydropyrazolo[4,3-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)NN=C2 AWFYPPSBLUWMFQ-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000004354 Hydroxyethyl cellulose Substances 0.000 description 1
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000004721 Polyphenylene oxide Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 229920001218 Pullulan Polymers 0.000 description 1
- 239000004373 Pullulan Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 235000019423 pullulan Nutrition 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002277 temperature effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Landscapes
- Compositions Of Macromolecular Compounds (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、タンパク質水溶液の濃
縮方法に関する。The present invention relates to a method for concentrating an aqueous protein solution.
【０００２】[0002]
【従来の技術】タンパク質は一般に変性し易いことか
ら、水溶液中のタンパク質を濃縮する方法としては、塩
析や有機溶媒沈殿、凍結乾燥などにより固体として濃縮
する方法、吸着を利用する方法、半透膜を用いる方法、
吸水性ゲルを用いる方法などが用いられている。しか
し、濃縮処理速度の点で満足できなかったり、立体構造
の変化やゲル化等が遅こり易いタンパク質については、
こうした方法の適用も困難な場合があるなどの問題があ
った。2. Description of the Related Art Since proteins are generally easily denatured, methods for concentrating proteins in an aqueous solution include a method of concentrating them as a solid by salting out, an organic solvent precipitation, freeze-drying, etc., a method using adsorption, a semipermeable method, and the like. A method using a membrane,
A method using a water-absorbing gel is used. However, for proteins that are unsatisfactory in terms of the concentration processing speed, or that are liable to delay in changes in three-dimensional structure or gelation,
There were problems such as the difficulty in applying such a method.
【０００３】特に、絹タンパク質フィブロインは高濃度
の塩溶液に溶解させた後、透析することにより水溶液と
することができる。この水溶液は、シルクペプチドの製
造原料としてばかりでなく、塩析や乾燥、攪拌（ズリ応
カ）等の処理が加えられると、フィブロインが部分的に
結晶化して不溶化する。この現象を利用して膜、粉末、
ゲルなどに成形することができることから、化粧品用の
絹粉末（特公昭５８−３８４４９）や、酵素や抗体等の
固定化膜の製造に有効に応用されている（特公昭５６−
３８１９３、特公昭６０−１３６７２、特公平４−３９
６２３）が、目的によってはこの水溶液を濃縮して用い
ることが多い。[0003] In particular, silk protein fibroin can be made into an aqueous solution by dissolving it in a high-concentration salt solution and then dialysis. This aqueous solution is not only used as a raw material for producing a silk peptide, but also undergoes treatment such as salting out, drying, and agitation (slipperiness), whereby fibroin partially crystallizes and becomes insoluble. Using this phenomenon, films, powders,
Since it can be formed into a gel or the like, it has been effectively applied to the production of silk powder for cosmetics (Japanese Patent Publication No. 58-38449) and an immobilized film of enzymes, antibodies, etc.
38193, JP-B-60-13672, 4-39
623), but the aqueous solution is often concentrated and used depending on the purpose.
【０００４】しかし、この水溶液は、上述したように容
易にゲル化したり、フィブロインの析出を招いたりする
ため、その濃縮に際してはこれらの点に十分配慮する必
要があった。一つの方法として、水の加熱留去により濃
縮することができるが、この場合も一部析出やゲル化が
おこるため、歩留まりの低下を招くばかりでなく、微少
なゲル状部はろ過によっても除去し難いことから、得ら
れた膜に粒状の不均一部を与えることがある。また、半
透膜を用いる方法のうち、限外ろ過法においては膜表面
の物質濃縮による効率低下を解消するため、膜内液を攪
拌する必要があるが、攪拌により加えられるズリ応力に
よりフィブロインの析出がおこる。また、タンパク質水
溶液を入れた透析膜表面に送風したり、ポリエチレング
リコール粉末を接触させる方法（Agr.Biol.Che.３０，
５７６−５８４，１９６６）も用いることができるが濃
縮効率、処理速度に問題があった。However, since this aqueous solution easily gels or causes precipitation of fibroin as described above, it is necessary to pay sufficient attention to these points when concentrating the aqueous solution. As one method, concentration can be performed by heating and distilling off water.However, in this case, precipitation or gelation occurs partially, which not only causes a decrease in yield, but also removes fine gel-like portions by filtration. It is difficult to perform the treatment, and the resulting film may have a granular nonuniform portion. In addition, among the methods using a semipermeable membrane, in the ultrafiltration method, it is necessary to agitate the liquid in the membrane in order to eliminate the efficiency decrease due to the concentration of the substance on the membrane surface. Precipitation occurs. Further, a method of blowing air onto the surface of a dialysis membrane containing an aqueous protein solution or contacting it with polyethylene glycol powder (Agr. Biol. Che. 30,
576-584, 1966) can be used, but there are problems with the concentration efficiency and the processing speed.
【０００５】[0005]
【発明が解決しようとする課題】本発明の課題は、立体
構造の変化やゲル化等の変性をおこし易いタンパク質の
水溶液の濃縮に適した高効率の方法を提供することであ
る。An object of the present invention is to provide a highly efficient method suitable for concentrating an aqueous solution of a protein which is liable to undergo denaturation such as change in steric structure or gelation.
【０００６】[0006]
【課題を解決するための手段】本発明は、タンパク質水
溶液を透析膜を介して液状のポリエーテル系高分子或い
はポリオール系高分子の水溶液に接触させることを特徴
とするタンパク質水溶液の濃縮方法であり、前述の課題
を解決するものである。The present invention is a method for concentrating an aqueous protein solution, which comprises contacting an aqueous protein solution with an aqueous solution of a liquid polyether polymer or polyol polymer via a dialysis membrane. This is to solve the above-mentioned problem.
【０００７】[0007]
【発明の実施の形態】以下、本発明を詳細に説明する。
本発明で使用するポリエーテル系高分子としては、エチ
レングリコール誘導体として、ポリエチレングリコー
ル、エチレングリコール−プロピレングリコール共重合
体などが用いられ、末端がメチル基、エチル基、プロピ
ル基、ブチル基等のアルキル基で置換されているもの、
或いはその他の誘導体も同様に用いられる。更にこれら
を混合して用いることもできる。エチレングリコール−
プロピレングリコール共重合体としては、ブロック重合
体、ランダム共重合体の何れであってもよいが、高分子
量でも液状になり易い点からランダム共重合体が更に好
ましい。BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
As the polyether polymer used in the present invention, as an ethylene glycol derivative, polyethylene glycol, ethylene glycol-propylene glycol copolymer or the like is used, and the terminal is alkyl such as methyl group, ethyl group, propyl group, butyl group, etc. Substituted with a group,
Alternatively, other derivatives are used as well. Further, these can be used in combination. Ethylene glycol
The propylene glycol copolymer may be either a block polymer or a random copolymer, but is more preferably a random copolymer because it easily becomes liquid even with a high molecular weight.
【０００８】また、本発明で使用するポリオール系高分
子としては、デキストラン、プルラン等の多糖類、ヒド
ロキシエチルセルロース、ヒドロキシプロピルセルロー
ス等の多糖類誘導体、及びポリビニルアルコールなどを
用いることができる。更に、これらのポリエーテル系高
分子やポリオール系高分子を適宜混合して用いることも
できる。As the polyol polymer used in the present invention, polysaccharides such as dextran and pullulan, polysaccharide derivatives such as hydroxyethylcellulose and hydroxypropylcellulose, and polyvinyl alcohol can be used. Furthermore, these polyether-based polymers and polyol-based polymers can be appropriately mixed and used.
【０００９】これらの外系に用いるポリエーテル高分子
或いはポリオール高分子の分子量については、小さいも
のであっても濃縮効果の面では使用できるが、分子量が
小さくなるに従いタンパク質水溶液中に混入する割合が
増加する傾向にある。この程度はポリエチレングリコー
ル等の高分子の構造によっても異なるが、これを抑制す
るため、一般に分子量が１５００以上のものを使用する
ことが好ましく、２０００以上のものが更に好ましい。Regarding the molecular weight of the polyether polymer or polyol polymer used in the external system, even if the molecular weight is small, it can be used in terms of the concentration effect, but as the molecular weight becomes smaller, the proportion mixed into the aqueous protein solution becomes smaller. It tends to increase. Although the degree varies depending on the structure of a polymer such as polyethylene glycol, it is generally preferable to use one having a molecular weight of 1500 or more, and more preferably 2000 or more, in order to suppress this.
【００１０】本発明の濃縮方法を用いた場合の濃縮効率
は、用いるポリエーテル系高分子やポリオール系高分子
の種類、濃度、温度、及び攪拌の有無などに依存する。
また、内系の目的となるタンパク質の物性や濃度にも左
右されるが、一般に濃度が低く、粘性が小さいほど濃縮
速度は大きい。外系に用いる高分子としては、高濃度の
水溶液とした際の粘性が小さく、効率の高い濃縮効果を
与える点で、ポリエチレングリコールやランダム型のエ
チレングリコール−プロピレングリコール共重合体及び
それらの誘導体が好適に用いられる。[0010] The concentration efficiency when the concentration method of the present invention is used depends on the type, concentration, temperature, presence or absence of stirring, and the like of the polyether polymer and the polyol polymer used.
In addition, although it depends on the physical properties and concentration of the target protein in the inner system, generally, the concentration is low and the viscosity is small, the concentration rate is high. As the polymer used for the external system, polyethylene glycol or a random type ethylene glycol-propylene glycol copolymer and derivatives thereof are low in viscosity when used as a high-concentration aqueous solution and provide a highly efficient concentration effect. It is preferably used.
【００１１】外系の高分子濃度としては、用いる高分子
の種類によっても異なるが、一般に、２０％以上で用い
ることができる。更に濃縮効率及び速度の点で、５０％
以上であることが好ましい。The concentration of the polymer in the external system varies depending on the type of the polymer used, but generally it can be used at 20% or more. Furthermore, in terms of concentration efficiency and speed, 50%
It is preferable that it is above.
【００１２】また、ポリエーテル系高分子の１つである
ポリエチレングリコールは分子量が大きくなるに従い融
点が高くなり、１０００以上のものは室温ではワックス
状から固体状となる。そこで濃縮を行うタンパク質が熱
変性を起こさない範囲内で、加温によりポリエチレング
リコールを融解させ液状として本発明を実施することが
できるが、平均分子量１５００〜２０００のポリエチレ
ングリコールを用いて５０℃以下の温度で行うか、又は
液状エチレングリコール−プロピレングリコール共重合
体と混合して行うことが好ましい。The melting point of polyethylene glycol, one of the polyether polymers, increases as the molecular weight increases, and those of 1,000 or more change from waxy to solid at room temperature. Therefore, within the range in which the protein to be concentrated does not undergo thermal denaturation, the present invention can be carried out as a liquid by melting polyethylene glycol by heating. It is preferable to carry out at a temperature or by mixing with a liquid ethylene glycol-propylene glycol copolymer.
【００１３】更に、エチレングリコーループロピレング
リコール共重合体、なかでもランダム共重合体は、室温
領域で液状であるため、変性し易いタンパク質の濃縮に
も利用できる点で本発明に最も好適に用いることができ
るが、濃縮効率の点から分子量１５００以上、エチレン
グリコール分率２０％以上のものを用いることが好まし
い。Furthermore, ethylene glycol-propylene glycol copolymers, especially random copolymers, are liquid in the room temperature range, and therefore can be used most preferably in the present invention because they can be used for concentrating proteins that are easily denatured. However, it is preferable to use those having a molecular weight of 1500 or more and an ethylene glycol fraction of 20% or more from the viewpoint of concentration efficiency.
【００１４】本発明を実施するに際し、濃縮速度を高め
効率向上を計るために透析膜の外系のポリエチレングリ
コール誘導体液を攪拌することが好ましい。また、変性
などの問題のない範囲で加温することは外系の粘性を低
下させるので、攪拌効果の点からは好ましく、目的に応
じて適切な条件を選ぶことができる。In practicing the present invention, it is preferable to agitate the polyethylene glycol derivative liquid external to the dialysis membrane in order to increase the concentration rate and improve the efficiency. Heating in a range where there is no problem such as denaturation lowers the viscosity of the external system, so it is preferable from the viewpoint of the stirring effect, and appropriate conditions can be selected according to the purpose.
【００１５】以下、実施例を挙げて本発明の効果を詳述
する。Hereinafter, the effects of the present invention will be described in detail with reference to examples.
【００１６】[0016]
【実施例１】生糸を精錬後、エタノールを混合した４５
重量％塩化カルシウム水溶液に溶解し、これを透析して
絹フィブロイン水溶液（５．０重量％）を得た。この１
０ｇをセルロース透析チュープ（三光純薬株式会社：サ
イズ１８／３２）に入れ、２９℃で攪拌下、分子量の異
なる各種ポリエチレングリコール（ＰＥＧ）の５０重量
％水溶液０．１Ｌ中に浸漬した。６０分間浸漬後、ＰＥ
Ｇ水溶被より透析チェーブを取り出し、濃縮率及び絹フ
ィブロイン溶液中へのＰＥＧ混入量を測定した。絹フィ
ブロイン水溶液の濃縮率ほ、浸漬前後の重重変化を測定
し、元のフィブロィン濃度に対する攪拌後の濃度倍率を
算出した。またＰＥＧ混入量は、絹フィブロイン水溶液
の屈折率測定結果と、濃縮率より算出された絹フィブロ
イン濃度に対応する屈析率との差分を混入ＰＥＧによる
ものとして求めた。表１の結果より明らかなように、Ｐ
ＥＧの分子量に拘らず濃縮率はほぼ一定の値を与えた
が、ＰＥＧ平均分子量が小さくなるに従い、ＰＥＧ混入
量が増大する傾向が認められる。また、絹フィブロイン
溶液中へのＰＥＧ混入量は、ＰＥＧ平均分子量が小さく
なるに従い減少し、特に分子量２０００以上ではその値
を非常に小さくすることができる。尚、対照実験として
平均分子量１０００のポリプロピレングリコール（ＰＰ
Ｇ）に対して、２５℃で１時間浸漬処理を行ったが、濃
縮率１．０５倍と殆ど濃縮効果は認められなかった。ま
た、平均分子量２００００のＰＥＧ粉末を透析チェープ
表面に付着せしめ、２５℃で１時間放置する方法で濃縮
実験を行った結果、濃縮率は１．２倍であり、本発明の
方法に比較して低い効果しか得られなかった。Example 1 Raw silk was refined and mixed with ethanol.
It was dissolved in an aqueous solution of calcium chloride by weight and dialyzed to obtain an aqueous solution of silk fibroin (5.0% by weight). This one
0 g was placed in a cellulose dialysis tube (Sanko Junyaku Co., Ltd .: size 18/32) and immersed in 0.1 L of a 50% by weight aqueous solution of various polyethylene glycols (PEG) having different molecular weights at 29 ° C. with stirring. After immersion for 60 minutes, PE
G The dialysis chamber was taken out from the aqueous solution, and the concentration ratio and the amount of PEG mixed into the silk fibroin solution were measured. The concentration ratio of the silk fibroin aqueous solution and the change in weight before and after immersion were measured, and the concentration ratio after stirring to the original fibroin concentration was calculated. The amount of PEG mixed was determined as the difference between the refractive index measurement result of the aqueous solution of silk fibroin and the refractive index corresponding to the silk fibroin concentration calculated from the concentration ratio, based on the mixed PEG. As is clear from the results in Table 1, P
Although the enrichment ratio was almost constant irrespective of the molecular weight of EG, there was a tendency for the amount of PEG to increase as the average molecular weight of PEG became smaller. In addition, the amount of PEG mixed into the silk fibroin solution decreases as the average PEG molecular weight decreases, and particularly when the molecular weight is 2,000 or more, the value can be extremely reduced. As a control experiment, polypropylene glycol having an average molecular weight of 1000 (PP
G) was subjected to an immersion treatment at 25 ° C. for 1 hour, but the concentration rate was 1.05 times, and almost no concentration effect was observed. Further, a PEG powder having an average molecular weight of 20,000 was adhered to the surface of the dialysis cheep, and a concentration experiment was performed by leaving it at 25 ° C. for 1 hour. Only a low effect was obtained.
【００１７】[0017]
【表１】 [Table 1]
【００１８】[0018]
【実施例２】実施例１と同様にして調製した絹フィブロ
イン水溶液（濃度５．５重量％）１０ｇをセルロース透
析チューブに入れ、これを表２に示す各種濃度のＰＥＧ
（分子量３０００）水溶液０．１Ｌ中に攪拌下、各温度
で１時間浸漬した後取り出し、実施例１と同様の方法で
濃縮率を測定した。表２に示す限り、ＰＥＧ濃度が高
く、温度が高いほど絹フィブロイン水溶液の濃縮率が大
きいことがわかる。ＰＥＧ濃度が２０％以下では濃縮効
率が低く実用的ではない。Example 2 10 g of an aqueous solution of silk fibroin (concentration: 5.5% by weight) prepared in the same manner as in Example 1 was placed in a cellulose dialysis tube.
It was immersed in 0.1 L of an aqueous solution (molecular weight: 3000) at each temperature for 1 hour with stirring and then taken out. The concentration was measured in the same manner as in Example 1. As shown in Table 2, it can be seen that the higher the PEG concentration and the higher the temperature, the higher the concentration of the silk fibroin aqueous solution. If the PEG concentration is 20% or less, the concentration efficiency is low and not practical.
【００１９】[0019]
【表２】 [Table 2]
【００２０】[0020]
【実施例３】実施例１と同様にして調整した絹フィブロ
イン水溶液（濃度４．９重量％）２０ｇをセルロース透
析チェープに入れ、これを５０重量％のＰＥＧ水溶液
（平均分子量６０００）２００ｍｌ中に攪拌下２３．５
℃で浸漬した後、経時的に取り出し、実施例１と同様の
方法で濃縮率を測定し、濃縮速度を求めた。結果を表３
に示す。Example 3 20 g of an aqueous silk fibroin solution (concentration: 4.9% by weight) prepared in the same manner as in Example 1 was placed in a cellulose dialysis cheep, and stirred in 200 ml of a 50% by weight aqueous PEG solution (average molecular weight: 6000). Lower 23.5
After immersion at ℃, it was taken out with time, the concentration rate was measured in the same manner as in Example 1, and the concentration rate was determined. Table 3 shows the results
Shown in
【００２１】[0021]
【表３】 [Table 3]
【００２２】[0022]
【実施例４】実施例１と同様にして調整した絹フィブロ
イン水溶液（濃度４．８重量％）１０ｇをセルロース透
析チュープに入れ、これを表４に示す各種水溶液１５０
ｍｌ中に攪拌下、２７．０℃で６０分間浸漬した後取り
出し、実施例１と同様の方法で濃縮率を測定した。結果
を表４に示すが、ＰＥＧまたはその誘導体を用いた系が
特に良好な濃縮効果を与えた。また、いずれも外系高分
子のフィブロイン水溶液への混入は見られなかった。エ
チレングリコール（ＥＧ）−プロピレングリコール（Ｐ
Ｇ）共重合体のＥＧ／ＰＧ比は、 1Ｈ−ＮＭＲにより決
定し、ＥＧモル分率を示した。Example 4 10 g of an aqueous solution of silk fibroin (concentration: 4.8% by weight) prepared in the same manner as in Example 1 was placed in a cellulose dialysis tube, and this was mixed with various aqueous solutions shown in Table 4.
After immersion in 2 ml at 27.0 ° C. for 60 minutes with stirring, the solution was taken out, and the concentration ratio was measured in the same manner as in Example 1. The results are shown in Table 4. The system using PEG or its derivative gave a particularly good concentration effect. In addition, no external polymer was mixed into the aqueous fibroin solution. Ethylene glycol (EG) -propylene glycol (P
G) The EG / PG ratio of the copolymer was determined by 1 H-NMR and showed the EG mole fraction.
【００２３】[0023]
【表４】 [Table 4]
【００２４】[0024]
【実施例５】５０重量％のＰＥＧ水溶液２００ｍｌ（平
均分子量３０００）中に、表５に示した各種タンパク質
水溶液２５ｍｌを入れたセルロース透析チェーブを浸漬
し、外液を攪拌下、各所定の温度で２０分問の濃縮処理
を行った後、濃縮率を測定した。但し、タンパク質濃度
は、２８０ｎｍの吸光度測定により求めた。表５より明
らかなように濃縮速度に対し大きな温度効果が認めれ
た。また、いずれもタンパク質水溶液へのＰＥＧの混入
は認められなかった。Example 5 A cellulose dialysis tube containing 25 ml of various protein aqueous solutions shown in Table 5 was immersed in 200 ml of a 50% by weight aqueous PEG solution (average molecular weight: 3000), and the external solution was stirred at a predetermined temperature while stirring. After performing the concentration treatment for 20 minutes, the concentration ratio was measured. However, the protein concentration was determined by measuring the absorbance at 280 nm. As is clear from Table 5, a large temperature effect was observed on the concentration rate. In addition, no PEG was mixed into the aqueous protein solution in any case.
【００２５】[0025]
【表５】 [Table 5]
【００２６】[0026]
【実施例６】実施例１と同様にして調整した絹フィブロ
イン水溶液（濃度５．３重量％）１０ｇを透析チェーブ
に入れ、これを各種の液状エチレングリコール−プロピ
レングリコール共重合体０．１Ｌ中に２４．５℃で１時
間浸漬した後取り出し、実施例１と同様の方法で濃縮率
を測定した。結果を表６に示す。エチレングリコール
（ＥＧ）−プロピレングリコール（ＰＧ）共重合体のＥ
Ｇ／ＰＧ比は、 1Ｈ−ＮＭＲにより決定し、ＥＧモル分
率を示した。表６の中で、ＥＧ／ＰＧ，Ｂはエチレング
リコール−プロピレングリコールのプロック共重合体、
ＥＧ／ＰＧ，Ｒはランダム共重合体を示す。表６の結果
より、液状のポリエチレングリコール誘導体を用いるこ
とにより、室温でも有効な濃縮ができる。また、ＰＥＧ
誘導体の混入量はいずれも０．３％以下であり、濃縮後
のフィブロイン水溶液にはゲル化部や析出部が全く見ら
れなかった。Ｎｏ．２の例では、濃縮が進み水がＥＧ／
ＰＧ系に移行するに従い、外系の粘性が増大したため、
濃縮率が他の例より小さくなったものと考えられる。Example 6 10 g of an aqueous solution of silk fibroin (concentration: 5.3% by weight) prepared in the same manner as in Example 1 was placed in a dialysis chamber, and this was added to 0.1 L of various liquid ethylene glycol-propylene glycol copolymers. After being immersed at 24.5 ° C. for 1 hour, it was taken out, and the concentration rate was measured in the same manner as in Example 1. Table 6 shows the results. E of ethylene glycol (EG) -propylene glycol (PG) copolymer
The G / PG ratio was determined by 1 H-NMR and indicated the EG mole fraction. In Table 6, EG / PG, B are ethylene glycol-propylene glycol block copolymers,
EG / PG, R indicates a random copolymer. From the results in Table 6, it is possible to perform effective concentration even at room temperature by using a liquid polyethylene glycol derivative. Also, PEG
The amount of the derivative mixed was 0.3% or less in each case, and no gelled or precipitated portion was found in the concentrated fibroin aqueous solution. No. In the example of 2, the concentration proceeds and the water is EG /
As the viscosity of the external system increased with the shift to the PG system,
It is considered that the concentration ratio was smaller than the other examples.
【００２７】[0027]
【表６】 [Table 6]
【００２８】[0028]
【実施例７】実施例１と同様にして調製した絹フィブロ
イン水溶被（濃度４．９重量％）１０ｇを透析チューブ
に入れ、これを平均分子量３０００のポリエチレングリ
コールとエチレングリコール−プロピレングリコールラ
ンダム共重合体（平均分子量５０００、ＥＧモル分率８
７％）とを重量比で１：１に混合した溶液中に浸漬し、
２５℃、攪拌条件下で濃縮を行った。実施例１と同様の
方法で経時的に濃縮率を測定した。結果を表７に示す。
約１時間で２倍の濃縮率を得ることができる。Example 7 10 g of an aqueous solution of silk fibroin (concentration: 4.9% by weight) prepared in the same manner as in Example 1 was placed in a dialysis tube, and this was mixed with polyethylene glycol having an average molecular weight of 3000 and ethylene glycol-propylene glycol random copolymer. Combined (average molecular weight 5000, EG mole fraction 8
7%) and 1: 1 by weight.
Concentration was performed at 25 ° C. under stirring conditions. The concentration rate was measured over time in the same manner as in Example 1. Table 7 shows the results.
A double enrichment rate can be obtained in about one hour.
【００２９】[0029]
【表７】 [Table 7]
【００３０】[0030]
【実施例８】実施例７と同じ濃縮系を用いて、表８に示
した各種タンパク質水溶液の濃縮試験を行った。温度２
２．５℃、１時間の濃縮処理を行った後、濃縮率を測定
した。 但し、タンパク質濃度は２８０ｎｍの吸光度測
定により求めた。表８より明らかなようにいずれも高い
濃縮効果が認められた。Example 8 Using the same concentration system as in Example 7, concentration tests of various protein aqueous solutions shown in Table 8 were performed. Temperature 2
After performing the concentration treatment at 2.5 ° C. for 1 hour, the concentration ratio was measured. However, the protein concentration was determined by measuring the absorbance at 280 nm. As is clear from Table 8, a high concentration effect was observed in all cases.
【００３１】[0031]
【表８】 [Table 8]
【００３２】[0032]
【発明の効果】本発明の濃縮方法によれば、タンパク質
水溶液をタンパク質の変性を招くことなく短時間に濃縮
でき、透析膜表面に送風したり、ポリエチレングリコー
ル粉末を付着せしめる方法に較ぺて優れた濃縮効率を与
えるものである。特に、タンパク質が絹フィブロインの
場合、水を加熱留去する方法のように分子量低下、部分
ゲル化や析出、更に歩留まり低下を招くことがなく、工
業的に有効な濃縮法を与えるのみならず、酵素固定化膜
などの絹膜の製造に対しても、欠陥のない均一性の高い
膜を調製できる絹水溶液原料を提供できるものである。According to the concentration method of the present invention, the aqueous protein solution can be concentrated in a short time without denaturing the protein, and is superior to the method of blowing air onto the dialysis membrane surface or attaching polyethylene glycol powder. It provides a high concentration efficiency. In particular, when the protein is silk fibroin, a decrease in molecular weight, partial gelation or precipitation as in the method of heating and distilling off water does not cause a decrease in yield, and not only gives an industrially effective concentration method, The present invention can also provide a raw material for a silk aqueous solution that can be used to prepare a highly uniform film without defects even in the production of a silk film such as an enzyme-immobilized film.
【手続補正書】[Procedure amendment]
【提出日】平成８年９月２５日[Submission date] September 25, 1996
【手続補正１】[Procedure amendment 1]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】発明の名称[Correction target item name] Name of invention
【補正方法】変更[Correction method] Change
【補正内容】[Correction contents]
【発明の名称】 タンパク質水溶液の濃縮方法[Title of the Invention] The method of concentrating a protein aqueous solution
───────────────────────────────────────────────────── フロントページの続き (72)発明者 杉本 雅俊 大阪府八尾市山城町４丁目２番６号 (72)発明者 中山 博 大阪府枚方市東山１丁目38番５号 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Masatoshi Sugimoto 4-6-6 Yamashiro-cho, Yao-shi, Osaka (72) Inventor Hiroshi Nakayama 1-35-5 Higashiyama, Hirakata-shi, Osaka
Claims (3)
エーテル系高分子或いはポリオール系高分子の水溶液に
接触させることを特徴とするタンパク質水溶液の濃縮方
法。1. A method for concentrating an aqueous protein solution, comprising contacting the aqueous protein solution with an aqueous solution of a polyether polymer or a polyol polymer via a dialysis membrane.
ある請求項１の濃縮方法。2. The method according to claim 1, wherein the protein is silk protein fibroin.
以上のポリエチレングリコールである請求項１の濃縮方
法。3. The polyether polymer has a molecular weight of 1500.
The method for concentrating according to claim 1, which is the above polyethylene glycol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21522496A JPH1036676A (en) | 1996-07-26 | 1996-07-26 | Method for concentrating aqueous protein solution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP21522496A JPH1036676A (en) | 1996-07-26 | 1996-07-26 | Method for concentrating aqueous protein solution |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH1036676A true JPH1036676A (en) | 1998-02-10 |
Family
ID=16668769
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP21522496A Pending JPH1036676A (en) | 1996-07-26 | 1996-07-26 | Method for concentrating aqueous protein solution |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH1036676A (en) |
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