JPH0292283A - Immobilization of enzyme - Google Patents
Immobilization of enzymeInfo
- Publication number
- JPH0292283A JPH0292283A JP24449388A JP24449388A JPH0292283A JP H0292283 A JPH0292283 A JP H0292283A JP 24449388 A JP24449388 A JP 24449388A JP 24449388 A JP24449388 A JP 24449388A JP H0292283 A JPH0292283 A JP H0292283A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- porous carrier
- enzymes
- water
- inorganic salt
- 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
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 55
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 55
- 239000000126 substance Substances 0.000 claims abstract description 18
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 16
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 14
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 12
- 230000003100 immobilizing effect Effects 0.000 claims abstract description 11
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 6
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 18
- 238000001179 sorption measurement Methods 0.000 abstract description 20
- 102000004882 Lipase Human genes 0.000 abstract description 5
- 239000004367 Lipase Substances 0.000 abstract description 5
- 108090001060 Lipase Proteins 0.000 abstract description 5
- 235000019421 lipase Nutrition 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 230000005484 gravity Effects 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract description 3
- 108010053835 Catalase Proteins 0.000 abstract description 2
- 102000016938 Catalase Human genes 0.000 abstract description 2
- 239000007853 buffer solution Substances 0.000 abstract 1
- 230000003247 decreasing effect Effects 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 45
- 239000003054 catalyst Substances 0.000 description 7
- 239000000969 carrier Substances 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 239000012051 hydrophobic carrier Substances 0.000 description 5
- 229920003169 water-soluble polymer Polymers 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
- 239000012044 organic layer Substances 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 229920000233 poly(alkylene oxides) Polymers 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- KXGFMDJXCMQABM-UHFFFAOYSA-N 2-methoxy-6-methylphenol Chemical compound [CH]OC1=CC=CC([CH])=C1O KXGFMDJXCMQABM-UHFFFAOYSA-N 0.000 description 1
- 101710172561 3alpha-hydroxysteroid dehydrogenase Proteins 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 102100024089 Aldo-keto reductase family 1 member C2 Human genes 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102000015790 Asparaginase Human genes 0.000 description 1
- 108010024976 Asparaginase Proteins 0.000 description 1
- 102100023927 Asparagine synthetase [glutamine-hydrolyzing] Human genes 0.000 description 1
- 108010070255 Aspartate-ammonia ligase Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010011689 Glycine transaminase Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 108700040099 Xylose isomerases Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 229960003272 asparaginase Drugs 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- -1 etc. Substances 0.000 description 1
- 229920006248 expandable polystyrene Polymers 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229920001568 phenolic resin Polymers 0.000 description 1
- 239000005011 phenolic resin Substances 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920006327 polystyrene foam Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、酵素を疎水性の多孔性担体に高い吸着率で吸
着固定化する方法に関するものであり、さらに詳しくは
、酵素を、無機塩及び水溶性高分子物質の両成分の存在
下において、疎水性の多孔性担体に高い吸着率で吸着固
定化する方法に関するものである。[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a method for adsorbing and immobilizing an enzyme on a hydrophobic porous carrier with a high adsorption rate. The present invention relates to a method of adsorbing and immobilizing a water-soluble polymer substance on a hydrophobic porous carrier at a high adsorption rate in the presence of both components.
酵素は、生体細胞が産生ずる一種の有機触媒であり、機
能上すなわち触媒としての根本的性質は、一般の化学触
媒と全く同じといえる。Enzymes are a type of organic catalyst produced by living cells, and their functionality, that is, their fundamental properties as catalysts, can be said to be exactly the same as general chemical catalysts.
しかしながら、酵素は、タンパク質を主要構成成分とす
る生物触媒としての特性から、実際に触媒として使用す
る場合の取扱い方式は、化学触媒の場合と大きな差異が
あり、通常、酵素は、水溶性の形態でいわゆる回分方式
により使用されるため、一般の化学触媒のように反復利
用することができないこと、また、−反応ごとに多量の
酵素が消費されること等、酵素には、工業上の利用性の
面での種々のマイナス要因が存在する。However, due to the characteristics of enzymes as biocatalysts whose main component is protein, the way they are handled when actually used as a catalyst differs greatly from that of chemical catalysts, and enzymes are usually used in water-soluble form. Since enzymes are used in a so-called batch method, they cannot be used repeatedly like general chemical catalysts, and large amounts of enzyme are consumed for each reaction. There are various negative factors in this respect.
そこで、酵素を一般の化学触媒と同様に取扱うことを可
能にする方法として、酵素をその活性を保持させたまま
不溶化したいわゆる不溶化酵素の形態で基質と反応させ
ようとするアイディアが提案され、酵素の基礎研究、工
業的利用の両面において、多くの利点を与える革新的技
術として、確立されるに至った。Therefore, as a method to make it possible to handle enzymes in the same way as general chemical catalysts, an idea was proposed to react with substrates in the form of so-called insolubilized enzymes, in which enzymes retain their activity. It has come to be established as an innovative technology that offers many advantages in both basic research and industrial use.
酵素の不溶化の方式としては、酵素タンパク分子そのも
のに疎水性の官能基を導入する方法、酵素を水不溶性の
担体と結合させる方法等が基本的なものであり、このう
ち、後者については、吸着力のある担体に酵素を直接吸
着させ、物理吸着により酵素を担体に結合させた不溶化
酵素すなわち吸着固定化酵素等が種々開発されている。The basic methods for insolubilizing enzymes include introducing a hydrophobic functional group into the enzyme protein molecule itself, and binding the enzyme to a water-insoluble carrier. Various types of insolubilized enzymes, that is, adsorption-immobilized enzymes, have been developed in which enzymes are directly adsorbed onto strong carriers and the enzymes are bonded to the carrier by physical adsorption.
例えば、活性炭、酸性白土等を吸着剤として使用し、こ
れらに、糖化型アミラーゼを吸着させて調製した水に不
溶なコンプレックスを用いてデンプンの加水分解を行っ
た事例等が報告されている（「工業化学雑誌」７２巻、
２号（１９６９）第４８９−４９２頁）。For example, it has been reported that starch was hydrolyzed using a water-insoluble complex prepared by adsorbing saccharified amylase using activated carbon, acid clay, etc. as an adsorbent (""Industrial Chemistry Magazine" Volume 72,
No. 2 (1969), pp. 489-492).
（発明が解決しようとする課題）
ところで、このような吸着担体に酵素を吸着させて調製
した吸着固定化酵素を、工業上利用可能な実用化技術と
して完成させるためには、実際問題として、越えるべき
い（つかの障壁が存在する。(Problems to be Solved by the Invention) However, in order to perfect adsorption-immobilized enzymes prepared by adsorbing enzymes onto such adsorption carriers as a practical technology that can be used industrially, as a practical matter, it is necessary to overcome (There are some barriers.)
その代表的なものとして、吸着担体に対する酵素の吸着
率の問題があげられる。従来、吸着固定化酵素について
、高い吸着率を達成する好適な吸着担体の開発、吸着率
を向上化させるための吸着条件の検討等、多角的な検討
が重ねられてきた結果、特に、疎水性担体に関しては、
例えば、無機塩を添加して酵素を吸着固定化する方法等
が開発されているが、本発明者らが検討したところでは
、従来の方法は、必ずしも、疎水性担体に対する酵素の
吸着率が、満足すべき値ではなく、これを工業上利用可
能なものとするには、これらに吸着率を一段と向上させ
る新しい技術を開発することが不可欠の前提条件となる
ことが判明した。A typical example of this is the problem of the adsorption rate of enzymes on adsorption carriers. Conventionally, as a result of repeated studies on adsorption-immobilized enzymes, including the development of suitable adsorption carriers that achieve high adsorption rates, and examination of adsorption conditions to improve adsorption rates, in particular, hydrophobic Regarding the carrier,
For example, methods have been developed in which enzymes are adsorbed and immobilized by adding inorganic salts, but the present inventors have investigated that conventional methods do not necessarily have a high adsorption rate of enzymes on hydrophobic carriers. It was found that the values were not satisfactory, and in order to make these values industrially usable, it was an essential prerequisite to develop a new technology to further improve the adsorption rate.
すなわち、酵素によっては高濃度の無機塩を添加しない
と疎水性担体に吸着されないこと及び高濃度の無機塩を
添加してもなお未吸着酵素が残ること等の問題点がある
ことがわかった。吸着率が低いことは一定量の酵素を吸
着させるのに、かさ比重の大きな多孔性担体の必要量が
増すことを意味するものであり、装置の大型化等の問題
が生じる。That is, it has been found that some enzymes have problems such as not being adsorbed onto a hydrophobic carrier unless a high concentration of inorganic salt is added, and unadsorbed enzymes remaining even after adding a high concentration of inorganic salt. A low adsorption rate means that the amount of porous carrier with a large bulk specific gravity required increases in order to adsorb a certain amount of enzyme, which causes problems such as an increase in the size of the device.
そこで、本発明者らは、吸着固定化酵素をめぐるこのよ
うな技術的背景を踏まえ、疎水性担体に対する酵素の吸
着率を一段と向上させる革新的な技術を開発することを
目標として鋭意検討を積み重ねた結果、高濃度の無機塩
と水溶性高分子物質の両成分の存在下で、酵素を疎水性
の多孔性担体に吸着固定化することにより、吸着率を一
段と向上化し得ることを見い出して本発明を完成するに
至った。Therefore, based on this technical background surrounding adsorbed and immobilized enzymes, the present inventors have conducted extensive studies with the goal of developing an innovative technology that will further improve the adsorption rate of enzymes to hydrophobic carriers. As a result, we discovered that the adsorption rate could be further improved by adsorbing and immobilizing enzymes on a hydrophobic porous carrier in the presence of both high-concentration inorganic salts and water-soluble polymeric substances. The invention was completed.
すなわち、本発明は、以下の通りである。That is, the present invention is as follows.
（１）無機塩及び水溶性高分子物質の両成分の存在下で
、酵素を疎水性の多孔性担体に吸着固定化することを特
徴とする酵素の固定化方法。(1) A method for immobilizing an enzyme, which comprises adsorbing and immobilizing an enzyme on a hydrophobic porous carrier in the presence of both an inorganic salt and a water-soluble polymeric substance.
（２）無機塩が、硫酸アンモニウムである上記（１）記
載の固定化方法。(2) The immobilization method according to (1) above, wherein the inorganic salt is ammonium sulfate.
（３）水溶性高分子物質が、ポリエチレングリコールで
ある上記（１）記載の固定化方法。(3) The immobilization method according to (1) above, wherein the water-soluble polymeric substance is polyethylene glycol.
（４）疎水性の多孔性担体が、活性炭である上記（１）
記載の固定化方法。(4) The above (1) in which the hydrophobic porous carrier is activated carbon.
Immobilization method as described.
なお、前記したように、無機塩を添加して酵素を疎水性
担体に吸着固定化する方法は公知であるが、無機塩と水
溶性高分子物質の両成分を添加すると酵素の疎水性担体
への吸着率が一段と向上することは、本発明者らが初め
て見い出した知見であり、このような事実についての報
告はこれまでに全く存在しない。As mentioned above, there is a known method for adsorbing and immobilizing enzymes on hydrophobic carriers by adding inorganic salts, but adding both the inorganic salts and water-soluble polymeric substances to the enzymes' hydrophobic carriers. The fact that the adsorption rate of is further improved is a finding discovered for the first time by the present inventors, and there have been no reports of this fact to date.
このように、本発明は、酵素を疎水性の多孔性担体に高
い吸着率で吸着固定化する方法を提供することを目的と
するものであり、その構成は、無機塩及び水溶性高分子
物質の両成分の存在下で、酵素を疎水性の多孔性担体に
吸着固定化することを特徴とするものである。As described above, the present invention aims to provide a method for adsorbing and immobilizing enzymes on a hydrophobic porous carrier with a high adsorption rate, and the composition is comprised of an inorganic salt and a water-soluble polymer substance. The enzyme is adsorbed and immobilized on a hydrophobic porous carrier in the presence of both components.
無機塩としては、硫酸アンモニウム、硝酸アンモニウム
、硝酸ナトリウム、塩化ナトリウム、塩化リチウム等の
一般的な無機塩が使用されるが、好適には硫酸アンモニ
ウムが使用される。As the inorganic salt, common inorganic salts such as ammonium sulfate, ammonium nitrate, sodium nitrate, sodium chloride, and lithium chloride are used, and ammonium sulfate is preferably used.
これらの無機塩の添加量は、水相の反応液に対して、飽
和度２５〜９５％が好ましいが、飽和度４０〜８０％が
特に好ましい。The amount of these inorganic salts added is preferably 25 to 95% saturation, particularly preferably 40 to 80% saturation, relative to the aqueous reaction solution.
水溶性高分子物質としては、ポリエチレングリコール、
ポリプロピレングリコール等のポリアルキレンオキシド
類、デキストラン等の多糖類、及びポリアクリルアミド
、ポリビニルアルコール等の合成ポリマー等が使用され
るが、特に好ましくはポリエチレングリコール等のポリ
アルキレンオキシド類が使用される。これらの水溶性高
分子物質の分子量としては、１，０００〜ｓｏｏ、 ｏ
ｏｏが好ましいが、特に好ましくは２，０００〜１０．
０００であり、その添加量は重量％で２．０〜６０％、
好ましくは２．５〜２０％である。Water-soluble polymer substances include polyethylene glycol,
Polyalkylene oxides such as polypropylene glycol, polysaccharides such as dextran, and synthetic polymers such as polyacrylamide and polyvinyl alcohol are used, and polyalkylene oxides such as polyethylene glycol are particularly preferably used. The molecular weight of these water-soluble polymer substances is 1,000 to soo, o
oo is preferred, particularly preferably 2,000 to 10.
000, and the amount added is 2.0 to 60% by weight,
Preferably it is 2.5 to 20%.
疎水性の多孔性担体としては、疎水性基を有するポリア
クリルアミドゲル、多ｔＩ！類ゲル、発泡ポリスチレン
ゲル、発泡フェノール樹脂ビーズ及び活性炭等であるが
、特に好ましくは活性炭が使用される。その添加量は、
特に制限はないが好ましくは１０％付近である。As a hydrophobic porous carrier, polyacrylamide gel having a hydrophobic group, multi-tI! These include gels such as polystyrene foam gel, foamed polystyrene gel, foamed phenolic resin beads, activated carbon, etc., and activated carbon is particularly preferably used. The amount added is
Although there is no particular limit, it is preferably around 10%.
ここで使用する酵素は、特に限定は無〈従来公知の各種
のものを単独で又は２種以上混合して使用することがで
きる。その代表例としては、カタラーゼ、グルコースオ
キシダーゼ等の酸化酵素類、グリシンアミノトランスフ
ェラーゼ等の転移酵素類、アスパラギナーゼ、リパーゼ
、プロテアーゼ等の加水分解酵素、グルコースイソメラ
ーゼ等の異性化酵素類、アスパラギンシンセターゼ等の
りガーゼ類及びアルコール脱水素酵素等の酸化還元酵素
類が例示される。これらのうち好ましいのは、加水分解
酵素や酸化還元酵素であり、特にリパーゼ（Ｐｏｒｃｉ
ｎｅ Ｐａｎｃｒｅａｓ由来）、３α−ヒドロキシステ
ロイド脱水素酵素が好適なものとしてあげられる。The enzymes used here are not particularly limited; various conventionally known enzymes can be used alone or in a mixture of two or more. Typical examples include oxidizing enzymes such as catalase and glucose oxidase, transferases such as glycine aminotransferase, hydrolytic enzymes such as asparaginase, lipase, and protease, isomerizing enzymes such as glucose isomerase, and glue enzymes such as asparagine synthetase. Examples include gauze and oxidoreductases such as alcohol dehydrogenase. Preferred among these are hydrolases and oxidoreductases, especially lipases (Porci).
ne Pancreas) and 3α-hydroxysteroid dehydrogenase are preferred.
〔発明の効果］
本発明は、無機塩と水溶性高分子物質の両成分の存在下
で、酵素を疎水性の多孔性担体に吸着固定化することに
より、吸着率を著しく向上化する効果を有する。それに
よって、かさ比重の大きな多孔性担体の使用が可能にな
ること、さらに、多孔性担体の使用量の減少化が可能に
なること、装置の小型化が可能となること等、工業上の
利用性の面でのメリットは、きわめて顕著なものである
。[Effects of the Invention] The present invention has the effect of significantly improving the adsorption rate by adsorbing and immobilizing enzymes on a hydrophobic porous carrier in the presence of both components, an inorganic salt and a water-soluble polymer substance. have As a result, it is possible to use porous carriers with a large bulk specific gravity, and furthermore, it is possible to reduce the amount of porous carriers used, and it is possible to miniaturize equipment, etc., for industrial applications. The sexual benefits are quite remarkable.
以下に、本発明の実施例及び比較例を記載して、さらに
本発明の詳細な説明する。EXAMPLES Below, Examples and Comparative Examples of the present invention will be described to further explain the present invention in detail.
実施例１〜３
所定量の酵素を、０．１Ｍりん酸緩衝液（ｐＨ７，０）
（１３μりに溶解させ、５０ｍＭＮ Ａ Ｄ Ｈ水溶液
（４，０μｌ）、飽和度９０％硫酸アンモニウム水溶液
（ｐＨ７，０） （３７μｍ）及びポリエチレングリコ
ール（Ｍ−６０００） （４，１■）を加え、撹拌した
。反応混合物にイソオクタン（０，２ｍｆ）及び活性炭
（１２ｍｇ） （粒状シラサギ、武田薬品工業■製）を
加え、−時間撹拌した。遠心分離により有機層を分離後
、水相から４μｌずつサンプリングして水層に残存して
いる未吸着酵素をローリ−法により定量した。結果を表
１に示す。Examples 1 to 3 A predetermined amount of enzyme was added to 0.1M phosphate buffer (pH 7.0)
(Dissolve the solution in 13μm, add 50mM N A D H aqueous solution (4.0μl), 90% saturation ammonium sulfate aqueous solution (pH 7.0) (37μm) and polyethylene glycol (M-6000) (4.1■), and stir. Isooctane (0.2 mf) and activated carbon (12 mg) (granular Shirasagi, Takeda Pharmaceutical Co., Ltd.) were added to the reaction mixture and stirred for - hours. After separating the organic layer by centrifugation, 4 μl each was sampled from the aqueous phase. The unadsorbed enzyme remaining in the aqueous layer was quantified by the Lowry method.The results are shown in Table 1.
但し、酵素としてリパーゼを用いた場合には、ＮＡＤＨ
は無添加である。However, when lipase is used as the enzyme, NADH
is additive-free.
比較例１〜４
所定量の酵素を、０．１Ｍりん酸緩衝液（ｐＨ７，０）
（１３μ！！、）に溶解させ、５０ｍＭＮ Ａ Ｄ Ｈ
水溶液（４，０μｌ）、飽和度９０％硫酸アンモニウム
水溶液（ｐＨ７，０） （３７μ！！、）を加え、撹拌
した。反応混合物にイソオクタン（０，２ｍｆ）及び活
性炭（１２ｍｇ）（粒状シラサギ、武田薬品工業■製）
を加え、−時間撹拌した。遠心分離により有機層を分離
後、水相から４μβずつサンプリングして水層に残存し
ている未吸着酵素をローリ−法により定量した。結果を
表１に示す。Comparative Examples 1 to 4 A predetermined amount of enzyme was added to 0.1M phosphate buffer (pH 7.0)
(13μ!!,) and 50mM N A D H
An aqueous solution (4.0 µl) and a 90% saturated ammonium sulfate aqueous solution (pH 7.0) (37 µl!!) were added and stirred. Iso-octane (0.2 mf) and activated carbon (12 mg) (granular Shirasagi, manufactured by Takeda Pharmaceutical Co., Ltd.) were added to the reaction mixture.
was added and stirred for - hour. After separating the organic layer by centrifugation, samples of 4 μβ were taken from the aqueous phase, and the amount of unadsorbed enzyme remaining in the aqueous layer was quantified by the Lowry method. The results are shown in Table 1.
但し、酵素としてリパーゼを用いた場合には、ＮＡＤＨ
は無添加である。However, when lipase is used as the enzyme, NADH
is additive-free.
（本頁以下余白）
１）特開昭６２−６９５９８号記［２（０，６■）２）
Ｐｏｒｃｉｎｅ Ｐａｎｃｒｅａｓ由来、ベーリンガ
ーマンハイム山之内社製（０，５■）
３）酵母由来、ベーリンガーマンハイム山之内社製（０
，５■）
４）飽和度６０％(Margins below this page) 1) Japanese Patent Application Laid-Open No. 62-69598 [2(0,6■)2)
Derived from Porcine Pancreas, manufactured by Boehringer Mannheim Yamanouchi (0,5■) 3) Derived from yeast, manufactured by Boehringer Mannheim Yamanouchi (0,5■)
, 5 ■) 4) Saturation degree 60%
Claims (3)
、酵素を疎水性の多孔性担体に吸着固定化することを特
徴とする酵素の固定化方法。(1) A method for immobilizing an enzyme, which comprises adsorbing and immobilizing an enzyme on a hydrophobic porous carrier in the presence of both an inorganic salt and a water-soluble polymeric substance.
の固定化方法。(2) The immobilization method according to claim 1, wherein the inorganic salt is ammonium sulfate.
ある請求項１記載の固定化方法。（４）疎水性の多孔性
担体が、活性炭である請求項１記載の固定化方法。(3) The immobilization method according to claim 1, wherein the water-soluble polymeric substance is polyethylene glycol. (4) The immobilization method according to claim 1, wherein the hydrophobic porous carrier is activated carbon.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24449388A JPH0292283A (en) | 1988-09-30 | 1988-09-30 | Immobilization of enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24449388A JPH0292283A (en) | 1988-09-30 | 1988-09-30 | Immobilization of enzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0292283A true JPH0292283A (en) | 1990-04-03 |
| JPH0417635B2 JPH0417635B2 (en) | 1992-03-26 |
Family
ID=17119488
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP24449388A Granted JPH0292283A (en) | 1988-09-30 | 1988-09-30 | Immobilization of enzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0292283A (en) |
-
1988
- 1988-09-30 JP JP24449388A patent/JPH0292283A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0417635B2 (en) | 1992-03-26 |
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