JPH01300894A - Immobilization of phosphorylase and purification thereof - Google Patents
Immobilization of phosphorylase and purification thereofInfo
- Publication number
- JPH01300894A JPH01300894A JP12997088A JP12997088A JPH01300894A JP H01300894 A JPH01300894 A JP H01300894A JP 12997088 A JP12997088 A JP 12997088A JP 12997088 A JP12997088 A JP 12997088A JP H01300894 A JPH01300894 A JP H01300894A
- Authority
- JP
- Japan
- Prior art keywords
- phosphorylase
- resin
- anion exchange
- exchange resin
- synthetic polymer
- 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
- 102000009097 Phosphorylases Human genes 0.000 title claims abstract description 46
- 108010073135 Phosphorylases Proteins 0.000 title claims abstract description 46
- 238000000746 purification Methods 0.000 title description 7
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 21
- 230000003100 immobilizing effect Effects 0.000 claims abstract description 13
- 229920001059 synthetic polymer Polymers 0.000 claims abstract description 12
- 125000001453 quaternary ammonium group Chemical group 0.000 claims abstract description 5
- 125000001302 tertiary amino group Chemical group 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 27
- 238000005349 anion exchange Methods 0.000 claims description 3
- 239000011347 resin Substances 0.000 abstract description 28
- 229920005989 resin Polymers 0.000 abstract description 28
- 230000000694 effects Effects 0.000 abstract description 14
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 abstract description 6
- 102000004190 Enzymes Human genes 0.000 abstract description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 4
- 238000001179 sorption measurement Methods 0.000 abstract description 3
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract description 2
- 238000005342 ion exchange Methods 0.000 abstract description 2
- 239000004925 Acrylic resin Substances 0.000 abstract 1
- 229920000178 Acrylic resin Polymers 0.000 abstract 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract 1
- 239000012535 impurity Substances 0.000 abstract 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 238000005406 washing Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 11
- 244000061456 Solanum tuberosum Species 0.000 description 10
- 235000002595 Solanum tuberosum Nutrition 0.000 description 10
- 238000010828 elution Methods 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 6
- 239000003456 ion exchange resin Substances 0.000 description 5
- 229920003303 ion-exchange polymer Polymers 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 4
- 229920000936 Agarose Polymers 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 229920002307 Dextran Polymers 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002952 polymeric resin Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- KAKZBPTYRLMSJV-UHFFFAOYSA-N Butadiene Chemical compound C=CC=C KAKZBPTYRLMSJV-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241001474791 Proboscis Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 235000014347 soups Nutrition 0.000 description 2
- CHRJZRDFSQHIFI-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;styrene Chemical compound C=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C CHRJZRDFSQHIFI-UHFFFAOYSA-N 0.000 description 1
- OHVLMTFVQDZYHP-UHFFFAOYSA-N 1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-2-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound N1N=NC=2CN(CCC=21)C(CN1CCN(CC1)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)=O OHVLMTFVQDZYHP-UHFFFAOYSA-N 0.000 description 1
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- HXXFSFRBOHSIMQ-VFUOTHLCSA-N alpha-D-glucose 1-phosphate Chemical compound OC[C@H]1O[C@H](OP(O)(O)=O)[C@H](O)[C@@H](O)[C@@H]1O HXXFSFRBOHSIMQ-VFUOTHLCSA-N 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 229950010772 glucose-1-phosphate Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 125000005496 phosphonium group Chemical group 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- RWSOTUBLDIXVET-UHFFFAOYSA-O sulfonium group Chemical group [SH3+] RWSOTUBLDIXVET-UHFFFAOYSA-O 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明はホスホリラーゼの固定化法及び精製法、更に詳
細には、固定化担体として合成高分子系アニオン交換樹
脂を使用するホスホリラーゼの固定化法及び精製法に関
する。[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a method for immobilizing and purifying phosphorylase, and more specifically, a method for immobilizing phosphorylase using a synthetic polymeric anion exchange resin as an immobilization carrier. and purification methods.
ホスホリラーゼは、例えば医薬用抗菌剤、抗ｍ瘍剤（白
金錯体）、心臓病の治ｆＩＩ楽（アミン塩）等として有
用なＰｓ糖系の初期化合物でめるグルコース−１−リン
酸（以下「Ｇ−１−Ｐ」と略称する）を会成する際に、
非常に有用な酵素でめり、これは馬鈴薯塊茎寺の植吻、
クサギ筋内等の動物、酵母などの微生物等に広く分布し
ている。Phosphorylase is an initial compound of the Ps sugar family, glucose-1-phosphate (hereinafter referred to as " G-1-P”),
It contains a very useful enzyme, which is the proboscis of the potato tuber temple,
It is widely distributed in animals, such as in the hamstring muscles, and in microorganisms such as yeast.
従来、ホスホリラーゼの固定化法としては、■セルロー
スにジエチルアミンエチル基を導入した弱塩基性アニオ
ン交換樹脂にイオン結合により固定する方法、■アガロ
ースにオクチル基を導入した樹脂に疎水結合により固定
する方法、及び■ＣＮＢｒで活性化されたアガロースに
共有結合によシ固定する方法が用いられている。Conventionally, methods for immobilizing phosphorylase include: (i) Immobilization by ionic bonding on a weakly basic anion exchange resin in which diethylamine ethyl groups have been introduced into cellulose; (ii) Immobilization in hydrophobic bonding in a resin in which octyl groups have been introduced into agarose; and (2) a method of covalently immobilizing it on agarose activated with CNBr.
しかしながら、これらの何れの方法も、ホスホリラーゼ
の吸着１ｔが低いため、Ｇ−１−Ｐ合成能力が低く、か
つ多Ｓ樹脂を使用するため樹脂の再生等の取シ扱いが難
しく、コストも制いという問題点がめった。However, in both of these methods, the G-1-P synthesis ability is low due to the low adsorption of 1t of phosphorylase, and the use of multi-S resin makes handling such as resin regeneration difficult, and costs are also limited. This problem occurred very often.
一万、ホスホリラーゼの′！ｆ！製法としては、ホスホ
リラーゼ含有液を、直接又は硫安塩析等の前処理を行っ
た後、上記のセルロース又はアガロースを母体とするイ
オン交換樹脂、あるいはデキストランを母体とするイオ
ン交換樹脂に吸着させ、溶出する方法が知られている。Ten thousand, phosphorylase'! f! The manufacturing method is to adsorb the phosphorylase-containing solution directly or after pretreatment such as ammonium sulfate salting out to the above-mentioned cellulose or agarose-based ion exchange resin, or dextran-based ion exchange resin, and elute it. There are known ways to do this.
すなわち、例えば、馬鈴薯の汁ｆ、４Ａ安で塩析した後
、デキストランを母体とするイオン交換樹脂に吸着させ
、溶出する方法（Ｊ。That is, for example, a method in which potato juice f is salted out with 4A ammonium, adsorbed onto an ion exchange resin containing dextran as a matrix, and eluted (J.
５ｔａｅｒｋら： Ｂｉｏｃｈｅｍ、 Ｂｉｏｐｈｙａ
、 Ａｃｔａ、 、 １４６ 。5taerk et al.: Biochem, Biophya
, Acta, , 146.
１２０（１９６７）Ｊ ；馬鈴薯の汁を熱処理した後
、デンゾン吸着、セルロース又はデキストランを母体と
するイオン交換ｗ脂に吸着させ、溶出する方法（Ａ、
Ｋａｍｏｇａｗａら： Ｊ、 Ｂｉｏｃｈｅｍ、。120 (1967) J; A method of heat-treating potato juice, adsorbing it to Denzon adsorption, ion exchange fat using cellulose or dextran as a matrix, and eluting it (A,
Kamogawa et al.: J. Biochem.
６３．３６１ （１９６８）］；家兎筋肉抽出液をアガ
ロースを母体とするイオン交換樹脂に吸着させ、溶出す
る方法（Ｅｒ−ｅｌ、Ｚら：Ｂｉｏｃｈｅｍ、 Ｂｉｏ
ｐｈｙｓ、 Ｒｅｇ、 ＣｏＭｒｌｕｎ、、 ４９ 。63.361 (1968)]; a method in which rabbit muscle extract is adsorbed to an ion exchange resin containing agarose as a matrix and eluted (Er-el, Z et al.: Biochem, Bio
phys, Reg, CoMrlun, 49.
３８３ （１９７２） ）等が報告されている。383 (1972)) etc. have been reported.
しかしながら、これらの方法は、多糖を母体とするイオ
ン交換樹脂を使用するため、物理的強度が弱い、流速を
上げ難い、体積変化が大きい、アルカリに弱い、リガン
ドをカッブリングするのに煩雑な操作を必要とする、コ
ストが高いと共に、多量の硫安を必要とし、全工程が煩
雑でろるという欠点かめシ、工業的方法として不利なる
を免れなかった。However, these methods use ion exchange resins that have polysaccharides as their base material, so they have weak physical strength, are difficult to increase the flow rate, have large volume changes, are sensitive to alkalis, and require complicated operations to couple the ligands. This method is disadvantageous as an industrial method because it is expensive, requires a large amount of ammonium sulfate, and the entire process is complicated and slow.
従って、従来から上記問題点を克服したホスホリラーゼ
の固定化及び精製法の開発が所望されていた。Therefore, it has been desired to develop a method for immobilizing and purifying phosphorylase that overcomes the above-mentioned problems.
斯かる実状において、本発明者らは鋭意研究を行った結
果、合成高分子糸アニオン交換樹脂がホスホリラーゼの
固定化担体として極めて優れていること、そして該樹脂
を使用すれば、工業的有利にホスホリラーゼの固定化及
び精製を行うことができることを見出し、本発明を完成
した。Under these circumstances, the present inventors have conducted intensive research and found that synthetic polymer thread anion exchange resin is extremely excellent as a support for immobilizing phosphorylase, and that the use of this resin can provide an industrially advantageous method for immobilizing phosphorylase. The present invention has been completed based on the discovery that it is possible to immobilize and purify .
すなわち、本発明は、ホスホリラーゼ含有溶液を合成高
分子系アニオン交換樹脂に接触させるととを特徴とする
ホスホリラーゼの固定化法、並びにホスホリラーゼ含有
溶液を脅威高分子系アニオン交換樹脂に接触させてホス
ホリラーゼを固定化し、次いでこれを溶出することを特
徴とするホスホリラーゼのｆｆＷ法を提供するものであ
る。That is, the present invention provides a phosphorylase immobilization method characterized by contacting a phosphorylase-containing solution with a synthetic polymer anion exchange resin, and a method for immobilizing phosphorylase by contacting a phosphorylase-containing solution with a synthetic polymer anion exchange resin. The present invention provides an ffW method for phosphorylase, which is characterized by immobilizing it and then eluting it.
本発明に用いるアニオン交換樹脂としては、例エバスチ
レン系、ビニル系、ゾロピレン系、エチレン系、ブタジ
ェン系、アクリロニトリル系、インクレン系、アクリル
酸・メタアクリル酸系、フェノール系、フェノール−ｍ
−フェニレンシアミン系、エビクルヒドリン系が挙げら
れ、その甲でも時にスチレン系、アクリル酸系の樹脂が
好ましい。アニオン交換基としては、第一級〜第三級ア
ミ７基、第四級アンモニウム基、ホスホニウム基、スル
ホニウム基等が挙げられ％荷に第三級アミン基、第四級
アンモニウム基が好ましい。交換容重粒径に関しては荷
に限定されないが、交換容量は０．０１〜ｌ Ｑ ｍｅ
ｑ ／−樹脂が好ましく、特に０．１〜３ ｍｅｑ ／
ｍｌ樹脂が好ましい。粒径は５０〜１０００μｍが好
ましい。Examples of the anion exchange resin used in the present invention include ebastyrene type, vinyl type, zolopyrene type, ethylene type, butadiene type, acrylonitrile type, increne type, acrylic acid/methacrylic acid type, phenol type, and phenol-m
-Phenylenecyamine-based and eviclhydrin-based resins are mentioned, and styrene-based and acrylic acid-based resins are sometimes preferred. Examples of the anion exchange group include primary to tertiary amine groups, quaternary ammonium groups, phosphonium groups, and sulfonium groups, with tertiary amine groups and quaternary ammonium groups being preferred. The exchange capacity of heavy particles is not limited to the load, but the exchange capacity is 0.01~l Q me
q/-resin is preferred, especially 0.1 to 3 meq/
ml resin is preferred. The particle size is preferably 50 to 1000 μm.
本発明方法は、固定化担体として曾成高分子系アニオン
交換樹脂を使用する以外は、ホスホリラーゼの公知の固
定化法及び精製法に従って実施される。The method of the present invention is carried out according to known immobilization and purification methods for phosphorylase, except that a synthetic polymer anion exchange resin is used as the immobilization carrier.
ホスホリラーゼの固定化は、例えば、活性化した又は活
性化した後緩衝溶液で平衡化した甘酸高分子系アニオン
交換樹脂に、動物、植物、微生物等から得たホスホリラ
ーゼ又はその含有物を接触させてホスホリラーゼを該樹
脂に吸着させ、次いでイオン交換水、クエン酸緩衝液等
の緩ｍ液で付層した不純切を洗浄することによって行わ
れる。Immobilization of phosphorylase can be carried out, for example, by contacting phosphorylase obtained from animals, plants, microorganisms, etc. or its contents with a sweet acid polymer anion exchange resin that has been activated or equilibrated with a buffer solution after activation. is adsorbed onto the resin, and then the impure particles formed in the layer are washed with a mild solution such as ion-exchanged water or citric acid buffer.
ホスホリラーゼ含Ｍ俗液と合成高分子系アニオン交換樹
脂との接触は、両者を混合し、攪拌又はａ盪する方法、
あるいは該樹脂金力２ムにつめ、これにホスホリラーゼ
含有溶液を流通させる方法によって行われる。ホスホリ
ラーゼ含有溶液に対する合成高分子系アニオン交換樹脂
の使用量は時に限定されないが、体積比で１．０以上、
ホスホリラーゼ全活性０に対して該アニオン交換樹脂５
．０（Ｕ／ｆ）以上を使用するのが好ましい。The contact between the phosphorylase-containing M solution and the synthetic polymer anion exchange resin can be carried out by mixing the two and stirring or agitating;
Alternatively, it can be carried out by filling the resin into a container and flowing a phosphorylase-containing solution through it. The amount of synthetic polymer anion exchange resin used in the phosphorylase-containing solution is not limited, but the volume ratio is 1.0 or more,
The anion exchange resin 5 for total phosphorylase activity 0
．． It is preferable to use 0 (U/f) or more.
ホスホリラーゼを精製するには、精製せんとするホスホ
リラーゼを上記の如くシて合成高分子系アニオン交換１
Ｍ脂に吸着させて固定化し、次いでこれからホスホリラ
ーゼに！出させればよい。To purify phosphorylase, the phosphorylase to be purified is purified using synthetic polymer anion exchange method 1 as described above.
It is adsorbed to M fat and immobilized, and then it becomes phosphorylase! Just let it come out.
溶出方法は脣に限定されず、例えば塩及びｐＨｉコント
ロールする一般的な方法が採用される。その中でも、０
．１〜２Ｍの塩化ナトリウム、特に０．１〜０．５Ｍの
塩化ナトリウム溶液、好ましくはクエンｒｌＲ緩衝液等
の緩衝液で調製した塩化ナトリウムｆｆＩ液を溶出液と
して使用するのがよい結果を与える。溶出はホスホリラ
ーゼ固定樹脂と溶出液とを混合して攪拌又は振盪する方
法、めるいはホスホリラーゼ固定樹脂をカラムにつめ溶
出液を流通する方法等によって行われる。溶出液の重は
ホスホリラーゼ固定樹脂に対し、体積比で１０以上が好
ましい。The elution method is not limited to the elution method, and for example, a general method of controlling salt and pHi may be employed. Among them, 0
．． Good results are obtained by using a sodium chloride ffI solution prepared in a buffer such as a 1-2M sodium chloride solution, especially a 0.1-0.5M sodium chloride solution, preferably a citrate rlR buffer, as the eluent. Elution is carried out by mixing the phosphorylase-immobilized resin and the eluate and stirring or shaking the mixture, or by packing the phosphorylase-immobilized resin in a column and passing the eluate through the column. The weight of the eluate is preferably 10 or more in volume ratio to the phosphorylase-immobilized resin.
ホスホリラーゼの固定化及び溶出の際の温度は１〜４０
℃、特に１〜５℃が好ましく、また攪拌は樹脂が壊れな
い程度の回転数で、振盪は１〜１５０ｓｐｍ、％に７０
〜１００ ａｐｍで、更にカラムへの流通は樹脂の耐圧
以下の流速で行うのが好ましい。その他の条件、すなわ
ち固定化時間、溶出時間、カラム流通時間及び添加剤、
防腐剤の添加等は目的に応じて設定すればよい。The temperature during immobilization and elution of phosphorylase was 1 to 40°C.
℃, especially preferably 1 to 5℃, stirring at a rotation speed that does not break the resin, shaking at 1 to 150 spm, and 70% to 70%.
~100 apm, and the flow to the column is preferably performed at a flow rate lower than the pressure resistance of the resin. Other conditions, i.e. immobilization time, elution time, column flow time and additives,
Addition of preservatives, etc. may be determined depending on the purpose.
本発明のホスホリラーゼの固定化法は、ホスホリラーゼ
を荷足のアニオン交ｍ樹脂に固定化するため、＃索を有
効に利用でき且つまた固定化樹脂への酵素液層重が高い
ため反応性が良く、固定化酵素の活性保持性が良好であ
ると共に、固定化担体でろるアニオン交換樹脂の物理的
強度が強いこと等からＧ−１−Ｐを安価に製造できるも
のである。The phosphorylase immobilization method of the present invention immobilizes phosphorylase on the anionic resin, which makes it possible to effectively utilize #strings, and also has good reactivity because the enzyme liquid layer weight on the immobilization resin is high. , G-1-P can be produced at a low cost because the immobilized enzyme has good activity retention properties and the anion exchange resin that dissolves on the immobilization carrier has strong physical strength.
また、本発明のホスホリラーゼの精製法は特定のアニオ
ン５！換樹脂に固定化し、溶出させるだけの簡単な操作
で行うことができ、かつ選択的な液層、溶出によシ回収
率が高く。In addition, the method for purifying phosphorylase of the present invention uses a specific anion 5! It can be carried out with a simple operation of immobilizing it on a resin and eluting it, and the recovery rate is high due to the selective liquid phase and elution.
精製用担体であるアニオン交換樹脂の物理的強度も強い
こと等からホスホリラーゼを安価に精製できるものであ
る。Phosphorylase can be purified at low cost because the anion exchange resin used as the purification carrier has strong physical strength.
以下に実施例を挙げて説明する。 Examples will be described below.
なお、ここで示す酵素活性ＩＵとは、３０℃で１分間に
１μｍｏｌの生ｋｌｔ吻を得るのに必要な酵素量と定義
する。Note that the enzyme activity IU shown here is defined as the amount of enzyme required to obtain 1 μmol of fresh klt proboscis per minute at 30°C.
馬鈴薯のすシ汁は、馬鈴薯をジューサーでつぶし、遠心
分離にかけて調製した。Potato sushi soup was prepared by crushing potatoes with a juicer and centrifuging them.
全活性回収率及び全タンノ９り回収率は、初期の馬鈴薯
のすシ汁の全活性、全タンノ９りに対する溶出液の全活
性、全タンノ９りの、ｅ−セントで定義した。また比活
性は、全活性を全タン、Ｑりで割った値でＵ ／ ｑの
単位を持ち、精製倍率は、溶出液の比活性を初期の馬鈴
薯のすり汁の比活性で割った値と定義した。The total activity recovery rate and the total recovery rate were defined as the total activity of the initial potato sushi juice, the total activity of the eluate relative to the total concentration, and the e-cent of the total concentration. In addition, specific activity is the value obtained by dividing the total activity by total tan and Q, and has the unit of U / q, and the purification ratio is the value obtained by dividing the specific activity of the eluate by the specific activity of the initial potato juice. defined.
実施例１
Ａ生したスチレン系（スチレンジビニルベンゼン）合成
高分子樹脂（粒径３８０μｍ）に第四級アンモニウム基
（トリエチルアミノエチル基）を導入したアニオン交換
樹脂（交換ｄ重０．６５ｍｅｑ／−樹脂）２０−に馬鈴
薯のすυ汁２００−を加え、２５．７℃ｓ８２ｓｐｍで
５時間撮盪した後水洗した。その結果２８、ＯＵ／ｒ樹
脂の固定化ホスホリラーゼが得られた。Example 1 Anion exchange resin (exchange d weight 0.65 meq/-resin) in which quaternary ammonium groups (triethylaminoethyl groups) were introduced into A-produced styrene-based (styrene divinylbenzene) synthetic polymer resin (particle size 380 μm) ) 20- was added with potato soup 200-, and the mixture was shaken at 25.7°C and 82 spm for 5 hours, and then washed with water. As a result, 28, immobilized phosphorylase on OU/r resin was obtained.
実施例２
再生した？リアクリル系合成高分子樹脂（粒径１２０μ
ｍ）に第三級アミン基（ゾエチ基
ルアミノエチルコを導入したアニオン交換樹脂（交換容
量０．１２ｍｅｑ／−樹脂）２０−に馬鈴薯のすシ汁２
００−を加え、２５．７℃、６２ ａｐｍで５時間振盪
した後水洗した。その結果、２１．６Ｕ／ｆ樹脂の固定
化ホスホリラーゼが得られた。Example 2 Played? Reacrylic synthetic polymer resin (particle size 120μ
Anion exchange resin (exchange capacity 0.12 meq/-resin) into which a tertiary amine group (zoethyl group ruaminoethylco) has been introduced into m) 20- potato sushi juice 2
00- was added thereto, and the mixture was shaken at 25.7°C and 62 apm for 5 hours, and then washed with water. As a result, immobilized phosphorylase of 21.6 U/f resin was obtained.
実施例３
再生した？リアクリル系甘酸高分子樹脂＜ｆｉ２径１２
０μｍ）に第三級アンモニウム基（ジエチルアミンエチ
ル基）を導入したアニオン又ＩＩ８樹脂（交換容量０．
１２ｍｅｑ／−樹脂）ｉ、 ｉ ９ ｔをカラムにつめ
、馬鈴薯のすシ汁＆９５１を流速１．７９Ｊ／ｈｒの流
速で流し、次いでクエン酸緩衝液で洗浄した。その結果
、３３．８ Ｕ／？樹脂の固定化ホスホリラーゼが得ら
れた。Example 3 Played? Reacrylic sweet acid polymer resin<fi2 diameter 12
Anion or II8 resin (exchange capacity 0.0 μm) with a tertiary ammonium group (diethylamine ethyl group) introduced
12 meq/-resin) i, i9t was packed into a column, potato sushi juice &951 was passed through the column at a flow rate of 1.79 J/hr, and then washed with citric acid buffer. As a result, 33.8 U/? Resin immobilized phosphorylase was obtained.
実施例４
実施例２で得た２ Ｌ６Ｕ／を樹脂の固定化ホスホリラ
ーゼ２０−に０．５ ＭのＮａ（４を２０〇−加え、２
５．７℃ｓ８２ｓｐｍで１時間振盪し溶出させた。その
結果、全活性回収率６５．９％％精製倍率７．３倍でホ
スホリラーゼがｒｌ製された。Example 4 2 L6U/ obtained in Example 2 was added to the immobilized phosphorylase 20- of resin and 0.5 M Na (200-
Elution was carried out by shaking at 5.7°C and 82 spm for 1 hour. As a result, phosphorylase was produced with a total activity recovery rate of 65.9% and a purification ratio of 7.3 times.
実施例５
実施例３で得た３３．８Ｕ／ｆ樹脂の固定化ホスホリラ
ーゼＬ１９１をカラムにつめ、０、５ Ｍ ）Ｎ＆Ｃ１
溶液１０６を流速１０ｊ／時の流速で流し、溶出させた
。その結果、全活性回収率７６．６％、精製倍率５．２
倍でホスホリラーゼが精製された。Example 5 Immobilized phosphorylase L191 of 33.8 U/f resin obtained in Example 3 was packed in a column, and 0.5 M) N&C1
Solution 106 was eluted by flowing at a flow rate of 10 j/hour. As a result, the total activity recovery rate was 76.6%, and the purification ratio was 5.2.
Phosphorylase was purified twice.
以上that's all
Claims (1)
換樹脂に接触させることを特徴とするホスホリラーゼの
固定化法。 ２、合成高分子系アニオン交換樹脂のアニオン交換基が
、第三級アミノ基又は第四級アンモニウム基である請求
項１記載のホスホリラーゼの固定化法。 ３、ホスホリラーゼ含有溶液を合成高分子糸アニオン交
換樹脂に接触させてホスホリラーゼを固定化し、次いで
これを溶出することを特徴とするホスホリラーゼの精製
法。[Claims] 1. A method for immobilizing phosphorylase, which comprises bringing a phosphorylase-containing solution into contact with a synthetic polymer anion exchange resin. 2. The method for immobilizing phosphorylase according to claim 1, wherein the anion exchange group of the synthetic polymeric anion exchange resin is a tertiary amino group or a quaternary ammonium group. 3. A method for purifying phosphorylase, which comprises bringing a phosphorylase-containing solution into contact with a synthetic polymer thread anion exchange resin to immobilize phosphorylase, and then eluting it.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12997088A JPH01300894A (en) | 1988-05-27 | 1988-05-27 | Immobilization of phosphorylase and purification thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12997088A JPH01300894A (en) | 1988-05-27 | 1988-05-27 | Immobilization of phosphorylase and purification thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01300894A true JPH01300894A (en) | 1989-12-05 |
Family
ID=15022937
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12997088A Pending JPH01300894A (en) | 1988-05-27 | 1988-05-27 | Immobilization of phosphorylase and purification thereof |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01300894A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5535110A (en) * | 1978-08-31 | 1980-03-12 | Honda Motor Co Ltd | Internal combustion engine equipped with semi-spherical type inner wall surface of combustion chamber |
| JPS61257184A (en) * | 1985-05-10 | 1986-11-14 | Tokuyama Soda Co Ltd | Carrier for immobilized enzyme |
-
1988
- 1988-05-27 JP JP12997088A patent/JPH01300894A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5535110A (en) * | 1978-08-31 | 1980-03-12 | Honda Motor Co Ltd | Internal combustion engine equipped with semi-spherical type inner wall surface of combustion chamber |
| JPS61257184A (en) * | 1985-05-10 | 1986-11-14 | Tokuyama Soda Co Ltd | Carrier for immobilized enzyme |
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