JPH06169772A - Immobilized biocatalyst and method for producing the same - Google Patents
Immobilized biocatalyst and method for producing the sameInfo
- Publication number
- JPH06169772A JPH06169772A JP32348792A JP32348792A JPH06169772A JP H06169772 A JPH06169772 A JP H06169772A JP 32348792 A JP32348792 A JP 32348792A JP 32348792 A JP32348792 A JP 32348792A JP H06169772 A JPH06169772 A JP H06169772A
- Authority
- JP
- Japan
- Prior art keywords
- strain
- microbial cell
- solution
- pseudomonas putida
- cultured
- 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
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 29
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 29
- 239000011942 biocatalyst Substances 0.000 title claims abstract description 20
- 238000004519 manufacturing process Methods 0.000 title claims description 10
- 241000589776 Pseudomonas putida Species 0.000 claims abstract description 12
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 210000004748 cultured cell Anatomy 0.000 claims description 4
- 239000000243 solution Substances 0.000 abstract description 13
- 241000588724 Escherichia coli Species 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 8
- 238000004132 cross linking Methods 0.000 abstract description 6
- 230000000813 microbial effect Effects 0.000 abstract description 6
- 239000000126 substance Substances 0.000 abstract description 6
- 239000006285 cell suspension Substances 0.000 abstract description 5
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 239000007853 buffer solution Substances 0.000 abstract description 2
- 238000009630 liquid culture Methods 0.000 abstract description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract 2
- 230000006866 deterioration Effects 0.000 abstract 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract 1
- 239000003054 catalyst Substances 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 19
- 230000001580 bacterial effect Effects 0.000 description 10
- 238000000034 method Methods 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 244000005700 microbiome Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 150000001733 carboxylic acid esters Chemical class 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 239000003431 cross linking reagent Substances 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 102220201851 rs143406017 Human genes 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 210000001822 immobilized cell Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- PUKLCKVOVCZYKF-UHFFFAOYSA-N 1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2,5-dione Chemical compound O=C1C=CC(=O)N1CCN1C(=O)C=CC1=O PUKLCKVOVCZYKF-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- OWIKHYCFFJSOEH-UHFFFAOYSA-N Isocyanic acid Chemical class N=C=O OWIKHYCFFJSOEH-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 101000616888 Pseudomonas putida Mandelate racemase Proteins 0.000 description 1
- 244000292604 Salvia columbariae Species 0.000 description 1
- 235000012377 Salvia columbariae var. columbariae Nutrition 0.000 description 1
- 235000001498 Salvia hispanica Nutrition 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
- 150000004753 Schiff bases Chemical class 0.000 description 1
- XUGUHTGSMPZQIW-UHFFFAOYSA-N [[4-(4-diazonioiminocyclohexa-2,5-dien-1-ylidene)cyclohexa-2,5-dien-1-ylidene]hydrazinylidene]azanide Chemical compound C1=CC(N=[N+]=[N-])=CC=C1C1=CC=C(N=[N+]=[N-])C=C1 XUGUHTGSMPZQIW-UHFFFAOYSA-N 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 230000002152 alkylating effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000014167 chia Nutrition 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920006254 polymer film Polymers 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明は、カルボン酸エステルを
不斉加水分解する反応に用いる固定化生体触媒に関す
る。TECHNICAL FIELD The present invention relates to an immobilized biocatalyst used in a reaction for asymmetrically hydrolyzing a carboxylic acid ester.
【0002】[0002]
【従来の技術】シュードモナス プチダ (Pseudomonas
putida) MR-2068 株及びエセリキアコリ (Escherichia
coli) MR-2103 株は、カルボン酸エステルを不斉加水分
解し、光学純度の高い光学活性カルボン酸を生成する酵
素を生産する微生物として提案されている (特開平1-22
2798号公報、特願平3-249923号参照) 。これらの微生物
が生産する酵素は耐熱性に優れており、産業上広い範囲
での応用が期待されている。[Prior Art] Pseudomonas
putida ) MR-2068 strain and Escherichia
coli ) MR-2103 strain has been proposed as a microorganism that produces an enzyme that asymmetrically hydrolyzes a carboxylic acid ester to produce an optically active carboxylic acid with high optical purity (JP-A 1-222).
(See Japanese Patent No. 2798 and Japanese Patent Application No. 3-249923). The enzymes produced by these microorganisms have excellent thermostability and are expected to be applied in a wide range of industries.
【0003】一方、固定化生体触媒応用技術は日進月歩
の進歩を遂げており、目的に応じて様々な固定化方法が
可能である。例えば、1) 水不溶性の担体に酵素を結合
させる方法、2) 酵素を2個又はそれ以上の官能基を持
った試薬で架橋する架橋法、3) 酵素を高分子ゲルの微
細な格子の中に包み込むか、半透膜性の高分子の皮膜に
よって被覆する包括法などである。微生物、オルガネラ
などの固定化についても本質的には酵素の固定化方法と
同様である。On the other hand, the technology for applying immobilized biocatalysts is advancing rapidly, and various immobilization methods are possible depending on the purpose. For example, 1) a method in which an enzyme is bound to a water-insoluble carrier, 2) a cross-linking method in which an enzyme is cross-linked with a reagent having two or more functional groups, and 3) an enzyme in a fine lattice of a polymer gel. The encapsulation method includes wrapping in or coating with a semipermeable polymer film. Immobilization of microorganisms, organelles, etc. is essentially the same as the method of immobilizing enzymes.
【0004】バイオリアクターにより有用物質を生産す
る場合、酵素反応終了液からの有用物質の精製におい
て、溶媒抽出、再結晶、カラムクロマトグラフィーによ
る精製などが通常行われる。これらの工程において菌体
から溶出した変性タンパク質の存在はしばしば問題とな
る。例えば、反応終了液から目的物を溶媒で抽出する
時、エマルジョンが生成し、抽出不能となることが多々
ある。これらの問題点を回避するため、タンパク質の溶
出を防止した固定化生体触媒の使用が有効である場合が
多い。When a useful substance is produced in a bioreactor, solvent extraction, recrystallization, purification by column chromatography and the like are usually carried out in the purification of the useful substance from the enzyme reaction completed liquid. The presence of denatured proteins eluted from the cells during these steps is often a problem. For example, when the target substance is extracted from the reaction-terminated liquid with a solvent, an emulsion is often formed, which makes extraction impossible. In order to avoid these problems, it is often effective to use an immobilized biocatalyst that prevents protein elution.
【0005】[0005]
【発明が解決しようとする課題】本発明は、シュードモ
ナス プチダ (Pseudomonas putida) MR-2068 株又はエ
セリキア コリ (Escherichia coli) MR-2103 株を用い
た有用物質の生産に用いる固定化生体触媒及びその製造
法を提供することを目的とする。DISCLOSURE OF THE INVENTION The present invention relates to an immobilized biocatalyst used for the production of a useful substance using Pseudomonas putida MR-2068 strain or Escherichia coli MR-2103 strain and its production. The purpose is to provide the law.
【0006】[0006]
【課題を解決するための手段】本発明の固定化生体触媒
は、シュードモナス プチダ (Pseudomonas putida)MR-
2068 株又はエセリキア コリ (Escherichia coli) MR-
2103 株の培養菌体を架橋化処理したことを特徴とする
ものである。更に、本発明の固定化生体触媒の製造法
は、シュードモナス プチダ (Pseudomonas putida) MR
-2068 株又はエセリキア コリ (Escherichia coli) MR
-2103株の培養菌体を、グルタルアルデヒドを用いて架
橋化処理することを特徴とするものである。Means for Solving the Problems] Immobilized biocatalyst of the present invention, Pseudomonas putida (Pseudomonas putida) MR-
2068 strain or Escherichia coli MR-
It is characterized in that the cultured bacterial cells of strain 2103 are crosslinked. Furthermore, the method for producing an immobilized biocatalyst of the present invention is a method for producing Pseudomonas putida MR.
-2068 strain or Escherichia coli MR
-2103 strain cultured cells are cross-linked with glutaraldehyde.
【0007】本発明に使用されるシュードモナス プチ
ダ (Pseudomonas putida) MR-2068株及びエセリキア
コリ (Escherichia coli) MR-2103 株は、工業技術院微
生物工業技術研究所に、それぞれ微工研条寄第3846
号及び微工研条寄第3835号として寄託されている。
本発明に使用されるシュードモナス プチダ (Pseudomo
nas putida) MR-2068株及びエセリキア コリ (Escheri
chia coli) MR-2103 株の培養は、液体培養でも固体培
養でも行うことができる。培地としては、微生物が通常
資化し得る炭素源、窒素源、ビタミン、ミネラルなどの
成分を適宜配合したものが用いられる。培養は、微生物
が生育可能である温度及びpHで行われるが、通常50℃以
下の温度で、pH2〜11の範囲で行われる。微生物の生育
を促進させるため、通気攪拌を行ってもよい。 Pseudomonas putida MR-2068 strain and Escherichia used in the present invention
Escherichia coli MR-2103 strains were distributed to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, respectively.
No. 3 and No. 3835 of the National Institute of Micro-Technology.
Pseudomonas putida for use in the present invention (Pseudomo
nas putida) MR-2068 strain and Eserikia coli (Escheri
The chia coli MR-2103 strain can be cultivated in either liquid culture or solid culture. As the medium, a medium in which components such as a carbon source, a nitrogen source, vitamins and minerals that can normally be assimilated by microorganisms are appropriately mixed is used. The culturing is carried out at a temperature and pH at which the microorganism can grow, but it is usually carried out at a temperature of 50 ° C. or lower and a pH range of 2-11. Aeration and agitation may be performed to promote the growth of microorganisms.
【0008】一方、本発明に使用できる架橋試薬は、特
にその種類が限定されるものではなく、シッフ塩基をつ
くるグルタルアルデヒド、ペプチド結合をするイソシア
ン酸誘導体、N, N'-エチレンビスマレイミド、ジアゾ
カップリングをするビスジアゾベンジジン、アルキル化
するN, N'-ポリメチレンビスヨードアセトアミドなど
が使用可能である。On the other hand, the cross-linking reagent which can be used in the present invention is not particularly limited in its kind, and glutaraldehyde which forms a Schiff base, an isocyanic acid derivative which forms a peptide bond, N, N'-ethylene bismaleimide, diazo. Coupling bisdiazobenzidine, alkylating N, N′-polymethylenebisiodoacetamide and the like can be used.
【0009】本発明の固定化生体触媒の製造に用いる生
体触媒の形態は、培養終了液、湿菌体、乾燥菌体(例え
ば凍結乾燥菌体)、噴霧乾燥菌体又は有機溶媒(例えば
アセトン、トルエン等)で処理した菌体を用いることが
できる。製造法としては、通常の方法が適用でき、例え
ば、培養した菌体をpH緩衝液又は生理食塩水に懸濁し、
これに架橋試薬を添加し、架橋化させて固定化生体触媒
とする方法が使用可能である。The form of the biocatalyst used in the production of the immobilized biocatalyst of the present invention is a culture-finished solution, wet cells, dry cells (eg freeze-dried cells), spray-dried cells or an organic solvent (eg acetone, Cells treated with toluene or the like) can be used. As the production method, a usual method can be applied, for example, the cultured cells are suspended in a pH buffer solution or physiological saline,
It is possible to use a method in which a crosslinking reagent is added to this to be crosslinked to obtain an immobilized biocatalyst.
【0010】架橋試薬としてグルタルアルデヒドを用い
る場合、その添加量は菌体乾燥重量に対して通常0.1〜
10.0% (重量%) の範囲で添加することができるが、0.
5 〜5.0 % (重量%) の範囲で添加することが望まし
い。架橋化処理温度は通常5〜70℃の範囲で可能である
が、60℃以上の温度で処理することにより菌体の同時殺
菌が可能であり、非耐熱酵素を失活させることも可能で
ある。When glutaraldehyde is used as the cross-linking reagent, the addition amount thereof is usually 0.1 to the dry weight of the cells.
It can be added in the range of 10.0% (wt%),
It is desirable to add in the range of 5 to 5.0% (wt%). The cross-linking treatment temperature is usually within the range of 5 to 70 ° C., but it is possible to simultaneously sterilize the cells by treating at a temperature of 60 ° C. or higher, and it is also possible to deactivate the non-thermostable enzyme. .
【0011】架橋化処理時間は通常10分から5時間の範
囲で可能であるが、1時間以上処理することが望まし
い。固定化生体触媒は溶液状態又は凍結乾燥などによる
乾燥状態で使用可能である。このように本発明の固体化
生体触媒は調製方法が極めて簡単であり、この固定化生
体触媒をバイオリアクターによる有用物質生産に用いた
場合、反応液中への酵素の流出を最小限に防ぐことがで
きるため、精製工程における操作性の大幅な向上が期待
できる。The cross-linking treatment time can be usually in the range of 10 minutes to 5 hours, but it is desirable to perform the treatment for 1 hour or more. The immobilized biocatalyst can be used in a solution state or a dried state such as freeze-dried. As described above, the solid-state biocatalyst of the present invention is extremely easy to prepare, and when this immobilized biocatalyst is used for the production of useful substances by a bioreactor, it is necessary to minimize the outflow of the enzyme into the reaction solution. Therefore, the operability in the purification process can be expected to be significantly improved.
【0012】[0012]
【実施例】以下、実施例により本発明を更に具体的に説
明するが、本発明の範囲はこれらの実施例に限定される
ものではない。The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.
【0013】[0013]
【実施例1】 (1)固定化生体触媒の調製 シュードモナス プチダ (Pseudomonas putida) MR-206
8 株 (微工研条寄第3846号) を肉エキス0.5 %、ペ
プトン0.75%、NaCl 0.25 %、グルコース0.5%、マル
トエキス0.15%からなる液体培地 (pH6.8) 100mlに植菌
し、30℃で1日間振とう培養を行った。培養終了後、培
養液を遠心分離し、得られた菌体の全量をイオン交換水
で洗浄した後、約10mlのM/20燐酸緩衝液に懸濁した。
この菌体懸濁液に菌体の乾燥重量に対して0〜5.0 %各
濃度となるようにグルタルアルデヒド溶液を添加し、60
℃に昇温後、約1時間攪拌した。得られた菌体懸濁液を
固定化菌体溶液とした。 (2)残存活性及びタンパク質溶出濃度の測定 M/20燐酸緩衝液20mlに固定化菌体16mgを含む固定化菌
体溶液を添加し、カルボン酸エステルの加水分解活性を
測定した。また、上清中の溶出タンパク質濃度を Folin
-Lowry法で測定した。結果を表1に示す。いずれも未処
理のものを100%とした。Example 1 (1) Preparation of immobilized biocatalyst Pseudomonas putida MR-206
8 strains (Mikoken Kenjoyori No. 3846) were inoculated into 100 ml of liquid medium (pH 6.8) consisting of 0.5% meat extract, 0.75% peptone, 0.25% NaCl, 0.5% glucose and 0.15% malt extract, and 30 Shaking culture was carried out at 0 ° C for 1 day. After the completion of the culture, the culture solution was centrifuged, the whole amount of the obtained bacterial cells was washed with ion-exchanged water, and then suspended in about 10 ml of M / 20 phosphate buffer.
A glutaraldehyde solution was added to this cell suspension to a concentration of 0 to 5.0% based on the dry weight of the cells, and 60%
After the temperature was raised to ° C, the mixture was stirred for about 1 hour. The obtained bacterial cell suspension was used as an immobilized bacterial cell solution. (2) Measurement of residual activity and protein elution concentration Immobilized cell solution containing 16 mg of immobilized cells was added to 20 ml of M / 20 phosphate buffer, and the hydrolysis activity of carboxylic acid ester was measured. In addition, the concentration of eluted protein in the supernatant
-Measured by the Lowry method. The results are shown in Table 1. In each case, the untreated one was set to 100%.
【0014】[0014]
【表1】 [Table 1]
【0015】[0015]
【実施例2】 (1)固定化生体触媒の調製 エセリキア コリ (Escherichia coli) MR-2103 株 (微
工研条寄第3835号) を肉エキス0.5 %、ペプトン1.
0 %、NaCl 0.5%を含む液体培地 (pH7.0) 100mlに植菌
し、37℃で1日間振とう培養を行った。培養終了後、培
養液を遠心分離し、得られた菌体の全量をイオン交換水
で洗浄した後、約10mlのM/20燐酸緩衝液に懸濁した。
この菌体懸濁液に菌体の乾燥重量に対して0〜5.0 %各
濃度となるようにグルタルアルデヒド溶液を添加し、60
℃に昇温後、約1時間攪拌した。得られた菌体懸濁液を
固定化菌体溶液とした。 (2)残存活性及びタンパク質溶出濃度の測定 M/20燐酸緩衝液20mlに固定化菌体16mgを含む固定化菌
体溶液を添加し、カルボン酸エステルの加水分解活性を
測定した。また、上清中の溶出タンパク質濃度を Folin
-Lowry法で測定した。結果を表2に示す。いずれも未処
理のものを100%とした。Example 2 (1) Preparation of Immobilized Biocatalyst Escherichia coli MR-2103 strain (Mikoken Kenjoyori No. 3835) was prepared with 0.5% meat extract and 1.
100 ml of a liquid medium (pH 7.0) containing 0% and 0.5% of NaCl was inoculated and shake culture was carried out at 37 ° C for 1 day. After the completion of the culture, the culture solution was centrifuged, the whole amount of the obtained bacterial cells was washed with ion-exchanged water, and then suspended in about 10 ml of M / 20 phosphate buffer.
A glutaraldehyde solution was added to this cell suspension to a concentration of 0 to 5.0% based on the dry weight of the cells, and 60%
After the temperature was raised to ° C, the mixture was stirred for about 1 hour. The obtained bacterial cell suspension was used as an immobilized bacterial cell solution. (2) Measurement of residual activity and protein elution concentration Immobilized bacterial cell solution containing 16 mg of immobilized bacterial cells was added to 20 ml of M / 20 phosphate buffer solution, and the hydrolysis activity of carboxylic acid ester was measured. In addition, the concentration of eluted protein in the supernatant
-Measured by the Lowry method. The results are shown in Table 2. In each case, the untreated one was set to 100%.
【0016】[0016]
【表2】 [Table 2]
【0017】[0017]
【発明の効果】本発明の固定化生体触媒は、菌体表面を
架橋処理されているため、酵素活性の低下が見られな
い。また、微生物菌体からのタンパク質溶出量も未処理
のものに比較して1/3 以下に低減することが可能であ
る。このため、本発明の固定化生体触媒をバイオリアク
ターによる有用物質の生産に用いた場合、精製工程にお
ける大幅な操作性の向上が期待できる。EFFECT OF THE INVENTION Since the immobilized biocatalyst of the present invention is subjected to a cross-linking treatment on the surface of bacterial cells, the enzyme activity does not decrease. In addition, the amount of protein eluted from microbial cells can be reduced to 1/3 or less compared to untreated proteins. Therefore, when the immobilized biocatalyst of the present invention is used for the production of a useful substance by a bioreactor, it is expected that the operability in the purification step will be greatly improved.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 FI 技術表示箇所 C12R 1:19) (C12P 41/00 C12R 1:40) (C12P 41/00 C12R 1:19) ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Internal reference number FI technical display location C12R 1:19) (C12P 41/00 C12R 1:40) (C12P 41/00 C12R 1:19)
Claims (2)
putida) MR-2068 株又はエセリキア コリ (Escherichi
a coli) MR-2103 株の培養菌体を架橋化処理したことを
特徴とする固定化生体触媒。1. Pseudomonas
putida ) MR-2068 strain or Escherichi coli ( Escherichi
a coli ) An immobilized biocatalyst characterized in that cultured cells of MR-2103 strain are cross-linked.
putida) MR-2068 株又はエセリキア コリ (Escherichi
a coli) MR-2103 株の培養菌体を、グルタルアルデヒド
を用いて架橋化処理することを特徴とする請求項1記載
の固定化生体触媒の製造法。2. Pseudomonas
putida ) MR-2068 strain or Escherichi coli ( Escherichi
The method for producing an immobilized biocatalyst according to claim 1, wherein the cultured cells of the a coli ) MR-2103 strain are crosslinked with glutaraldehyde.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP32348792A JPH06169772A (en) | 1992-12-02 | 1992-12-02 | Immobilized biocatalyst and method for producing the same |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP32348792A JPH06169772A (en) | 1992-12-02 | 1992-12-02 | Immobilized biocatalyst and method for producing the same |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH06169772A true JPH06169772A (en) | 1994-06-21 |
Family
ID=18155242
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP32348792A Pending JPH06169772A (en) | 1992-12-02 | 1992-12-02 | Immobilized biocatalyst and method for producing the same |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH06169772A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6673582B2 (en) | 2000-01-25 | 2004-01-06 | Regents Of The University Of Minnesota | Microbes and methods for remediation |
US6825001B2 (en) | 1997-01-17 | 2004-11-30 | Regents Of The University Of Minnesota | DNA molecules and protein displaying improved triazine compound degrading ability |
-
1992
- 1992-12-02 JP JP32348792A patent/JPH06169772A/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6825001B2 (en) | 1997-01-17 | 2004-11-30 | Regents Of The University Of Minnesota | DNA molecules and protein displaying improved triazine compound degrading ability |
US6673582B2 (en) | 2000-01-25 | 2004-01-06 | Regents Of The University Of Minnesota | Microbes and methods for remediation |
US6919199B2 (en) | 2000-01-25 | 2005-07-19 | Regents Of The University Of Minnesota | Microbes and methods for remediation |
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