JPH0394679A - Immobilized microorganismic cell and its production - Google Patents
Immobilized microorganismic cell and its productionInfo
- Publication number
- JPH0394679A JPH0394679A JP23145389A JP23145389A JPH0394679A JP H0394679 A JPH0394679 A JP H0394679A JP 23145389 A JP23145389 A JP 23145389A JP 23145389 A JP23145389 A JP 23145389A JP H0394679 A JPH0394679 A JP H0394679A
- Authority
- JP
- Japan
- Prior art keywords
- cell
- microorganismic
- bacterial cells
- water
- immobilized
- 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
- 238000004519 manufacturing process Methods 0.000 title claims description 5
- 235000010443 alginic acid Nutrition 0.000 claims abstract description 26
- 229920000615 alginic acid Polymers 0.000 claims abstract description 26
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 239000003960 organic solvent Substances 0.000 claims abstract description 12
- 229920001296 polysiloxane Polymers 0.000 claims abstract description 12
- 239000011159 matrix material Substances 0.000 claims abstract description 10
- 229920005573 silicon-containing polymer Polymers 0.000 claims abstract description 10
- 239000007791 liquid phase Substances 0.000 claims abstract description 9
- 239000002131 composite material Substances 0.000 claims abstract description 7
- -1 alginic acid ion Chemical class 0.000 claims abstract description 6
- 239000003349 gelling agent Substances 0.000 claims abstract description 5
- 230000001580 bacterial effect Effects 0.000 claims description 34
- 229940072056 alginate Drugs 0.000 claims description 14
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000012429 reaction media Substances 0.000 claims description 8
- 230000000379 polymerizing effect Effects 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 15
- 239000000783 alginic acid Substances 0.000 abstract description 12
- 229960001126 alginic acid Drugs 0.000 abstract description 12
- 150000004781 alginic acids Chemical class 0.000 abstract description 11
- 239000000203 mixture Substances 0.000 abstract description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 abstract description 3
- 239000001110 calcium chloride Substances 0.000 abstract description 3
- 229910001628 calcium chloride Inorganic materials 0.000 abstract description 3
- 230000000717 retained effect Effects 0.000 abstract description 2
- 239000000499 gel Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 241000187654 Nocardia Species 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 2
- KWKAKUADMBZCLK-UHFFFAOYSA-N 1-octene Chemical compound CCCCCCC=C KWKAKUADMBZCLK-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 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 2
- 239000008272 agar Substances 0.000 description 2
- 235000010419 agar Nutrition 0.000 description 2
- 150000001336 alkenes Chemical class 0.000 description 2
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium trichloride Chemical compound Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 2
- 239000012431 aqueous reaction media Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- DCAYPVUWAIABOU-UHFFFAOYSA-N hexadecane Chemical compound CCCCCCCCCCCCCCCC DCAYPVUWAIABOU-UHFFFAOYSA-N 0.000 description 2
- 229930195733 hydrocarbon Natural products 0.000 description 2
- 150000002430 hydrocarbons Chemical class 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000000661 sodium alginate Substances 0.000 description 2
- 235000010413 sodium alginate Nutrition 0.000 description 2
- 229940005550 sodium alginate Drugs 0.000 description 2
- NJWSNNWLBMSXQR-UHFFFAOYSA-N 2-hexyloxirane Chemical compound CCCCCCC1CO1 NJWSNNWLBMSXQR-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000004996 alkyl benzenes Chemical class 0.000 description 1
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- WDIHJSXYQDMJHN-UHFFFAOYSA-L barium chloride Chemical compound [Cl-].[Cl-].[Ba+2] WDIHJSXYQDMJHN-UHFFFAOYSA-L 0.000 description 1
- 229910001626 barium chloride Inorganic materials 0.000 description 1
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 description 1
- 239000001639 calcium acetate Substances 0.000 description 1
- 229960005147 calcium acetate Drugs 0.000 description 1
- 235000011092 calcium acetate Nutrition 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000000679 carrageenan Substances 0.000 description 1
- 235000010418 carrageenan Nutrition 0.000 description 1
- 229920001525 carrageenan Polymers 0.000 description 1
- 229940113118 carrageenan Drugs 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000003822 epoxy resin Substances 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- LNEPOXFFQSENCJ-UHFFFAOYSA-N haloperidol Chemical compound C1CC(O)(C=2C=CC(Cl)=CC=2)CCN1CCCC(=O)C1=CC=C(F)C=C1 LNEPOXFFQSENCJ-UHFFFAOYSA-N 0.000 description 1
- 229920001600 hydrophobic polymer Polymers 0.000 description 1
- 238000006459 hydrosilylation reaction Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N n-Octanol Natural products CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 description 1
- 229920005615 natural polymer Polymers 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 1
- 239000003791 organic solvent mixture Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920000647 polyepoxide Polymers 0.000 description 1
- 239000002685 polymerization catalyst Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 229920002379 silicone rubber Polymers 0.000 description 1
- 239000004945 silicone rubber Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 229910001631 strontium chloride Inorganic materials 0.000 description 1
- AHBGXTDRMVNFER-UHFFFAOYSA-L strontium dichloride Chemical compound [Cl-].[Cl-].[Sr+2] AHBGXTDRMVNFER-UHFFFAOYSA-L 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 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、水一有機溶媒からなる二液相系で水難溶性基
質に微生物を作用させて反応生成物を採取する際の微生
物を担体に固定化した固定化菌体及びその製造方法に関
する。[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to a method in which microorganisms are used as a carrier when a reaction product is collected by acting on a poorly water-soluble substrate in a two-liquid phase system consisting of water and an organic solvent. The present invention relates to an immobilized microbial cell and a method for producing the same.
炭化水素等の水難溶性基質に微生物を作用させ、反応生
成物を採取する方法が種々行われている。この種の反応
は、反応効率を高めるために、往々にして、前記基質と
異なる有機溶剤を添加した、いわゆる水一有機溶媒から
なる二液相系からなる溶媒中で行われることがある（特
公昭６３−１３６７８号、同６３−１４９５１号公報参
照）。Various methods have been used to collect reaction products by allowing microorganisms to act on poorly water-soluble substrates such as hydrocarbons. In order to increase reaction efficiency, this type of reaction is often carried out in a solvent consisting of a two-liquid phase system consisting of water and organic solvent, to which an organic solvent different from the substrate is added (especially (See Publications No. 63-13678 and No. 63-14951).
一方、最近生戊物と菌体との分離を容易にするために、
微生物を固定化した、いわゆる固定化菌体が種々提案さ
れている。この固定化法として、菌体を包み込むような
状態で高分子ゲルに固定化する包括法がある。この方法
は、菌体の漏出が少なく、菌体と担体との化学結合がな
いので菌体の活性低下が少ない等の利点があるため、広
く採用されている。この包括固定化法で使用される担体
としては、アルギン酸、寒天、カラギーナン、キトサン
、ゼラチン、アルブミン等の天然高分子、アクリルアミ
ド、光架橋性樹脂、ポリウレタン、ポリビニルアルコー
ル、エポキシ樹脂、シリコーンゴム等の合成高分子が知
られている。On the other hand, recently, in order to facilitate the separation of the fungus and the fungal body,
Various so-called immobilized microbial cells in which microorganisms are immobilized have been proposed. As this immobilization method, there is an enveloping method in which the bacterial cells are immobilized in a polymer gel in a manner that envelops them. This method has been widely adopted because it has advantages such as less leakage of bacterial cells and less decrease in bacterial activity because there is no chemical bond between the bacterial cells and the carrier. Supports used in this entrapping immobilization method include natural polymers such as alginic acid, agar, carrageenan, chitosan, gelatin, and albumin, acrylamide, photocrosslinkable resins, polyurethane, polyvinyl alcohol, epoxy resins, and synthesized silicone rubber. polymers are known.
しかしながら、かかる担体を用いる方法は、水一有機溶
媒の二液相系で水難溶性基質を反応させる場合、担体の
素材が親水性の場合は、基質の透過性が悪く、また疎水
性の場合は、無機栄養成分の透過性が悪く、反応活性を
十分に高く保つことができず、反応収率が悪いという問
題があった。However, when using a method using such a carrier, when a poorly water-soluble substrate is reacted in a two-liquid phase system of water and an organic solvent, if the carrier material is hydrophilic, the permeability of the substrate is poor; However, there were problems in that the permeability of inorganic nutritional components was poor, the reaction activity could not be kept sufficiently high, and the reaction yield was poor.
本発明者は、上記問題を解決すべく、鋭意研究を進めた
結果、疎水性のポリマーであるシリコーンボリマーと親
水性のゲルであるアルキン酸ゲルの複合マトリックス中
に菌体を包括することにより、反応活性を高くできるこ
とがわかった。本発明は、かかる知見に基づいてなされ
たもので、本発明の目的は水一有機溶媒の二液相系で水
難溶性基質を反応させる場合でも、反応活性を十分に高
く保ち、反応効率を極めて高くできる固定化菌体及びそ
の製造方法を提供することにある。In order to solve the above problem, the present inventor conducted intensive research and found that by enclosing bacterial cells in a composite matrix of silicone polymer, which is a hydrophobic polymer, and alkynoic acid gel, which is a hydrophilic gel. It was found that the reaction activity could be increased. The present invention was made based on this knowledge, and the purpose of the present invention is to maintain sufficiently high reaction activity and to achieve extremely high reaction efficiency even when a poorly water-soluble substrate is reacted in a two-liquid phase system of water and an organic solvent. An object of the present invention is to provide a highly efficient immobilized bacterial cell and a method for producing the same.
本発明の固定化菌体は、シリコーンボリマーとアルギン
酸ゲルとの複合マトリックス中に菌体を分散保持させた
ことからなるもので、特に好ましくは、前記アルギン酸
ゲルに反応培地を含ませてなるものである。また、本発
明の固定化菌体の製造方法としては、菌体を、シリコー
ンブレボリマーとアルギン酸イオンとを含有する溶液に
加えた後、アルギン酸ゲル化剤と接触させるとともに、
当該シリコーンプレポリマーを重合させることからなる
ものである。The immobilized bacterial cells of the present invention consist of bacterial cells dispersed and retained in a composite matrix of silicone polymer and alginate gel, and particularly preferably, the immobilized bacterial cells contain a reaction medium in the alginate gel. It is. Furthermore, the method for producing immobilized bacterial cells of the present invention includes adding bacterial cells to a solution containing silicone brevolimer and alginate ions, and then contacting the cells with an alginate gelling agent.
It consists of polymerizing the silicone prepolymer.
本発明は、例えば、ノルマルパラフィンやオレフィン等
の炭化水素を、キャンデイダ属、ノカルディア属、ロド
コッカス属、コリネバクテリウム属、アルスロバクター
属、マイコバクテリウム属、シュードモナス属等の微生
物を用い、パラフィン、オレフイン、ハロゲン化パラフ
ィン、アルキルベンゼン等の水不溶性の有機溶媒と水と
の混合溶媒中で、酸化してカルボン酸やエボキサイドを
生成させる、水一有機溶媒の二液相系での水難溶性基質
の菌体による反応に適用される。また、ノカルディア属
、アスペルギルス属等の微生物菌体による各種のステロ
イド類の変換反応等にも適用できる。The present invention, for example, converts hydrocarbons such as normal paraffins and olefins into paraffin by using microorganisms such as Candida, Nocardia, Rhodococcus, Corynebacterium, Arthrobacter, Mycobacterium, and Pseudomonas. In a mixed solvent of water and a water-insoluble organic solvent such as olefin, halogenated paraffin, or alkylbenzene, the substrate is oxidized to produce carboxylic acid or eboxide in a two-liquid phase system of water and organic solvent. Applies to reactions caused by bacterial cells. It can also be applied to conversion reactions of various steroids by microorganisms such as Nocardia and Aspergillus.
シリコーンポリマーは、ジメチルシロキサンを構成単位
とするものであるが、本発明の適用に当たっては、特に
、制限はないが、スベイヤ−（Ｓｐｅｉｅｒ）触媒によ
るジメチルシロキサンプレポリマーのヒドロシリル化反
応による重合硬化を利用するものが、室温の比較的温和
な条件で、副反応なく進行するため、特に、好適である
。The silicone polymer has dimethylsiloxane as a constituent unit, and there are no particular restrictions on the application of the present invention, but polymerization and curing by a hydrosilylation reaction of a dimethylsiloxane prepolymer using a Speier catalyst may be used. This is particularly preferred because it proceeds under relatively mild conditions at room temperature without side reactions.
このボリマーは、市販品を用いると簡便で良い。It is convenient to use a commercially available polymer as this polymer.
一方、アルギン酸は、アルギン酸のナトリウム、カリウ
ム、アンモニウム塩を用いることができる。これは、塩
化カルシウム、酢酸カルシウム、硫酸アルミニウム、塩
化アルミニウム、塩化バリウム、塩化ストロンチウム等
の水溶液からなるゲル化剤と接触させることによりゲル
化する。On the other hand, as alginic acid, sodium, potassium, and ammonium salts of alginic acid can be used. This is gelled by contact with a gelling agent consisting of an aqueous solution of calcium chloride, calcium acetate, aluminum sulfate, aluminum chloride, barium chloride, strontium chloride, or the like.
尚、このアルギン酸ゲルには、反応培地を含ませること
が好ましい。この場合、アルギン酸ソーダを反応培地水
溶液に添加して、用いると良い。尚、この反応培地の量
は、当該固定化菌体中に１〜２０重量％とすることが好
ましい。Note that this alginate gel preferably contains a reaction medium. In this case, it is preferable to add sodium alginate to the aqueous reaction medium solution. Note that the amount of this reaction medium is preferably 1 to 20% by weight in the immobilized bacterial cells.
このシリコーンポリマーとアルギン酸ゲルのマトリック
スは、シリコーン１００％からアルギン酸１００％まで
の全範囲で、任意の割合のマトリックスが得られるが、
シリコーンとアルギン酸との比を３＝７〜７：３の範囲
とすることが好ましい。アルギン酸の割合を３以下とす
ると、固定化菌体の親水性が乏しくなり、また、アルギ
ン酸が７以上となると、逆に疎水性に乏しくなるため、
あまり好ましくない。This matrix of silicone polymer and alginate gel can be obtained in any proportion over the entire range from 100% silicone to 100% alginic acid.
The ratio of silicone to alginic acid is preferably in the range of 3=7 to 7:3. When the ratio of alginic acid is 3 or less, the hydrophilicity of the immobilized bacterial cells becomes poor, and when the ratio of alginic acid is 7 or more, the hydrophobicity becomes poor.
I don't like it very much.
このようなマトリックス中に固定化する菌体は、ｌ〜２
０重量％とすることが好ましい６１重量％以下であれば
、反応効率が悪く、また、２０重量％以上とすると基質
および生威物の拡散律速もしくは生成物阻害等の弊害が
でるため好ましくない。The number of bacterial cells immobilized in such a matrix is 1 to 2
If it is less than 61% by weight, which is preferably 0% by weight, the reaction efficiency will be poor, and if it is more than 20% by weight, problems such as rate-limiting diffusion of substrates and biochemicals or inhibition of products will occur, which is not preferable.
本発明の固定化菌体は、遠心分離などにより濃縮した菌
体のペーストを、シリコーンプレポリマーと０．５〜３
重量％濃度のアルギン酸塩を含む水溶液（当該水溶液と
して、反応培地水溶液を用いると好ましい）の混合物中
に加え、十分に撹拌することにより、菌体を良く分散さ
せ、この混合液を、ゲル化剤中に注射器等を用いて滴状
で押し出すことにより、アルギン酸をゲル化させるとと
もに、シリコーンプレポリマーを重合硬化させて、ビー
ズ状の標品として得ることができる。尚，この際、必要
に応じて、前記シリコーンプレポリマーの重合触媒を添
加して、当該ボリマーの硬化時間を短縮しても良い。さ
らに、このマトリックスは、シート状に成形しても良い
ことは云うまでもない。The immobilized bacterial cells of the present invention are prepared by mixing a paste of bacterial cells concentrated by centrifugation with a silicone prepolymer of 0.5 to 3
Add it to a mixture of an aqueous solution (preferably a reaction medium aqueous solution is used as the aqueous solution) containing alginate at a concentration of % by weight, stir well to disperse the bacterial cells well, and add this mixture to the gelling agent. By extruding it in droplets using a syringe or the like, the alginic acid is gelled, and the silicone prepolymer is polymerized and cured to obtain a bead-shaped specimen. At this time, if necessary, a polymerization catalyst for the silicone prepolymer may be added to shorten the curing time of the polymer. Furthermore, it goes without saying that this matrix may be formed into a sheet shape.
このようにして得られる固定化菌体は、バイオリアクタ
ー中に充填され、当該リアクターに基質と反応培地であ
る水一有機溶媒混合液もしくは、基質を含む有機溶媒単
一液及び空気が供給されて、反応が行われる。The immobilized bacterial cells obtained in this way are filled into a bioreactor, and the reactor is supplied with a substrate and a reaction medium, either a water-organic solvent mixture or a single organic solvent containing the substrate, and air. , the reaction takes place.
［実施例］
星生立斑星
保存培地上のノカルディアコラリーナＢ−２７６（微工
研菌寄第４０９４号）の菌体一白金耳を、グルコース１
％、Ｎｕｔｒｉｅｎｔ Ｂｒｏｔｈ Ｎｏ．２を２．５
％含む寒天培地上に植菌し、２４時間培養した。[Example] One platinum loop of Nocardia coralina B-276 (Feikoken Bokuyori No. 4094) on Hoshii Tachiboshi preservation medium was mixed with glucose.
%, Nutrient Broth No. 2 to 2.5
The cells were inoculated onto an agar medium containing 5% and cultured for 24 hours.
次いで、増殖した全菌体を１００ｍｌのＮＢＧ培地（水
道水ｌＱ当たり、グルコース１ ０ｇ， Ｎｕ乞ｒｉｅ
ｎｔ Ｂｒｏｔｈ Ｎｏ．２ ２ ５ｇ）に加え、温度
３０℃、１ ４ ０ｓｔｒｏｋｅｓ／ｍｉｎの振盪条件
下に、１７時間培養した。さらに、この培地から１０ｍ
ｌを抜き出し、新しいＮＢＧ培地１００ｍｌに加え、同
条件下４８時間振盪培養した。続いて、菌体を遠心分離
により集菌した後、リン酸緩衝液（ＰＨ８）及び反応培
地（精製水ＩＣ当り、Ｋ，ＨＰ０， １．７４ｇ；Ｍ
ｇＳＯ４・７Ｈ，０ １．５ｇ；ＦｅＳＯ４・７Ｈ，Ｏ
Ｏ．０ ５ｇ；ｐＨ８）で洗浄した。Next, all the grown bacterial cells were mixed with 100 ml of NBG medium (10 g of glucose per 1Q of tap water,
nt Broth No. 225g) and cultured for 17 hours at a temperature of 30°C and shaking at 140 strokes/min. Furthermore, 10 m from this medium
1 was extracted, added to 100 ml of fresh NBG medium, and cultured with shaking under the same conditions for 48 hours. Subsequently, after collecting the bacterial cells by centrifugation, phosphate buffer (PH8) and reaction medium (K, HP0, 1.74 g per IC of purified water; M
gSO4・7H,0 1.5g; FeSO4・7H,O
O. 0.5 g; pH 8).
舅』１亙幌』Ｌ匿
上記菌体懸濁液から遠心分離により水分を除去して菌体
ペーストを調製し、この１．７ｍｌ（菌体質量０ ．
３ ｇ−ＤＣＷ）を、市販のジメチルシロキサンの二液
性プレポリマー（トーレ・シリコーン（株）製，ＣＹ５
２−１１１，初期粘度１ ５ ０ ０ｃ，ｐ，ｓ）Ａ液
及びＢ液各４ｇ、計８ｇと、上記と同じ組或の反応培地
を含む２．５重量％濃度のアルギン酸ナトリウム水溶液
１０ｍｌとの混合液に加え、十分にかき混ぜた後、４０
℃に保温した０．１モル濃度の塩化カルシウムを含む反
応培地水溶液２ ０ ０ｍｌ中に注射器により滴状で押
出し、一晩撹拌下に放置することにより、シリコーンポ
リマー約５０％及びアルギン酸ゲル約５０％の複合マト
リクス中に包括された、粒径２〜５ｍｍのビーズ状の固
定化菌体約２０ｍｌを得た。Water was removed from the above bacterial cell suspension by centrifugation to prepare a bacterial cell paste, and 1.7 ml of this (1.7 ml of bacterial cell mass 0.
3 g-DCW) was mixed with a commercially available two-component dimethylsiloxane prepolymer (manufactured by Toray Silicone Co., Ltd., CY5).
2-111, initial viscosity 1500 c, p, s) 4 g each of liquid A and liquid B, total 8 g, and 10 ml of a 2.5 wt % sodium alginate aqueous solution containing the same reaction medium as above. Add to the mixture and stir thoroughly, then add 40
About 50% silicone polymer and about 50% alginate gel were extruded dropwise with a syringe into 200 ml of an aqueous reaction medium solution containing calcium chloride at a molar concentration of 0.1° C. and left under stirring overnight. Approximately 20 ml of bead-shaped immobilized bacterial cells with a particle size of 2 to 5 mm were obtained, which were encapsulated in a composite matrix.
さらに、同様の手順によって，シリコーンボリマー１０
０％からアルギン酸ゲルｌＯＯ％までの両者の混合割合
を種々変えたものについても、各々、上記と同量の菌体
を包括した固定化標品約２０ｍｌを得た。Furthermore, by the same procedure, silicone polymer 10
Approximately 20 ml of immobilized specimens containing the same amount of bacterial cells as above were obtained in each case in which the mixing ratio of both was varied from 0% to lOO% of alginate gel.
反一息
上記方法で得られた固定化菌体を２０ｍｌ、１−オクテ
ン４ｍｌおよびｎ−ヘキサデカン４０ｍｌを、それぞれ
坂口フラスコに入れ、３０℃で、１４０ ｓｔｒｏｋｅ
ｓ／ｍｉｎの往復振盪培養機で培養し、所定時間ごとに
サンプリングを行い、１，２−エポキシオクタンの濃度
を、ガスクロマトグラフィーにより分析した。この結果
を、次表に示した。20 ml of the immobilized bacterial cells obtained by the above method, 4 ml of 1-octene, and 40 ml of n-hexadecane were placed in a Sakaguchi flask, and heated at 30°C with 140 strokes.
The cells were cultured in a reciprocating shaking incubator at s/min, sampling was performed at predetermined time intervals, and the concentration of 1,2-epoxyoctane was analyzed by gas chromatography. The results are shown in the table below.
表
［発明の効果］
以上のように本発明は、シリコーンボリマーとアルギン
酸ゲルとの複合マトリックス中に菌体を分散保持させる
ので、水一有機溶媒の二液相系で水難溶性基質を反応さ
せる場合でも、反応活性を十分に高く保ち、反応効率を
極めて高くできるという格別の効果を有するものである
。Table [Effects of the Invention] As described above, the present invention allows bacterial cells to be dispersed and held in a composite matrix of silicone polymer and alginate gel, so that a poorly water-soluble substrate can be reacted in a two-liquid phase system of water and organic solvent. It has the special effect of keeping the reaction activity sufficiently high and making the reaction efficiency extremely high even in the case of a reaction.
Claims (3)
せる固定化菌体において、シリコーンポリマーとアルギ
ン酸ゲルとの複合マトリックス中に菌体を分散保持させ
たことを特徴とする固定化菌体(1) Immobilization of bacterial cells that is applied to a poorly water-soluble substrate using a two-liquid phase system of water and an organic solvent, characterized in that the bacterial cells are dispersed and held in a composite matrix of silicone polymer and alginate gel. bacterial body
せたことを特徴とする固定化菌体。(2) An immobilized bacterial cell, characterized in that the alginate gel according to claim (1) contains a reaction medium.
オンとを含有する溶液に加えた後、アルギン酸ゲル化剤
と接触させるとともに、当該シリコーンプレポリマーを
重合させることを特徴とする固定化菌体の製造方法。(3) Production of immobilized bacterial cells characterized by adding bacterial cells to a solution containing a silicone prepolymer and alginate ions, and then contacting with an alginate gelling agent and polymerizing the silicone prepolymer. Method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23145389A JP2717449B2 (en) | 1989-09-08 | 1989-09-08 | Immobilized cells and method for producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP23145389A JP2717449B2 (en) | 1989-09-08 | 1989-09-08 | Immobilized cells and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0394679A true JPH0394679A (en) | 1991-04-19 |
| JP2717449B2 JP2717449B2 (en) | 1998-02-18 |
Family
ID=16923755
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP23145389A Expired - Lifetime JP2717449B2 (en) | 1989-09-08 | 1989-09-08 | Immobilized cells and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2717449B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999011676A1 (en) * | 1997-08-29 | 1999-03-11 | Smithkline Beecham Plc | Formulation |
-
1989
- 1989-09-08 JP JP23145389A patent/JP2717449B2/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999011676A1 (en) * | 1997-08-29 | 1999-03-11 | Smithkline Beecham Plc | Formulation |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2717449B2 (en) | 1998-02-18 |
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