JPH0688A - Method for reducing carbonyl compound with immobilized microorganism - Google Patents
Method for reducing carbonyl compound with immobilized microorganismInfo
- Publication number
- JPH0688A JPH0688A JP18464392A JP18464392A JPH0688A JP H0688 A JPH0688 A JP H0688A JP 18464392 A JP18464392 A JP 18464392A JP 18464392 A JP18464392 A JP 18464392A JP H0688 A JPH0688 A JP H0688A
- Authority
- JP
- Japan
- Prior art keywords
- carbonyl compound
- immobilized
- carrier
- organic solvent
- microorganism
- 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
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明は固定化微生物を用いるカ
ルボニル化合物の還元方法に関する。FIELD OF THE INVENTION The present invention relates to a method for reducing carbonyl compounds using immobilized microorganisms.
【0002】[0002]
【従来の技術とその課題】従来より、カルボニル化合物
の化学的還元によつて、工業上重要な2級アルコール類
が大量に製造されている。これら化学的還元法として
は、水素化アルミニウムリチウム、水素化ホウ素リチウ
ムなどの触媒による方法や白金等の貴金属触媒を用いた
接触還元法などがあるが、触媒コストの問題や位置選択
性、立体選択性に劣る、製造法の安全性が完全には保て
ない等の問題があり、また、得られる生産物の収率も必
ずしも高くない。2. Description of the Related Art Conventionally, industrially important secondary alcohols have been produced in large quantities by chemical reduction of carbonyl compounds. These chemical reduction methods include a method using a catalyst such as lithium aluminum hydride and lithium borohydride and a catalytic reduction method using a noble metal catalyst such as platinum. However, there are problems of catalyst cost, regioselectivity, and stereoselection. There are problems such as poor productivity and inability to completely maintain the safety of the production method, and the yield of the obtained product is not necessarily high.
【0003】一方、特に光学活性なアルコールの合成を
目的として、位置選択性および立体選択性に優れる生体
触媒、例えばパン酵母や各種微生物の酸化還元酵素を用
いたカルボニル化合物の還元反応も広く試みられており
[藤沢有ら、有合化、44巻519(1986)参
照]、優れた成績を与える場合も知られている。しかし
ながら、生体触媒による還元反応では、用いる酵素ある
いは微生物菌体量が多量であり、反応後の生体触媒の除
去、生物的還元反応に要求される補酵素の供給等コスト
的な問題点がある。On the other hand, particularly for the purpose of synthesizing optically active alcohols, biocatalysts having excellent regioselectivity and stereoselectivity, for example, reduction reactions of carbonyl compounds using oxidoreductases of baker's yeast and various microorganisms have been widely tried. [Fujisawa, et al., Yuka, Vol. 44, 519 (1986)], and is known to give excellent results. However, in the reduction reaction by the biocatalyst, the amount of enzyme or microbial cells used is large, and there are cost problems such as removal of the biocatalyst after the reaction and supply of coenzyme required for the biological reduction reaction.
【0004】また、基質たるカルボニル化合物及び/又
は生産物たるアルコール類が水に不溶性もしくは難溶性
である場合には、基質を水中に強制分散させるために強
力な撹拌力が要求される。反応速度が低い、反応後の生
体触媒と生産物との分離が困難である、連続生産や繰り
返し生産が不可能である等の問題点がある。When the carbonyl compound as a substrate and / or the alcohol as a product is insoluble or sparingly soluble in water, strong stirring force is required to forcibly disperse the substrate in water. There are problems that the reaction rate is low, it is difficult to separate the biocatalyst and the product after the reaction, and continuous production or repeated production is impossible.
【0005】また、基質たるカルボニル化合物及び/又
は生産物たるアルコール類が水に不溶性もしくは難溶性
である場合には、基質を水中に強制分散させるために強
力な撹拌力が要求される、反応速度が低い、反応後の生
体触媒と生産物との分離が困難である、連続生産や繰り
返し生産が不可能である等の問題点がある。When the substrate carbonyl compound and / or the product alcohol is insoluble or sparingly soluble in water, a strong stirring force is required to forcibly disperse the substrate in water. Is low, it is difficult to separate the biocatalyst and the product after the reaction, continuous production or repeated production is impossible, and the like.
【0006】さらに、基質たるカルボニル化合物や生産
物たるアルコール類は酵素や微生物に対して毒性を発現
する場合が多く、従つて、反応系への基質添加量や生産
物の収量が非常に低くなり、また、生体触媒の長期安定
性が保てない等の問題点がある。Further, carbonyl compounds as substrates and alcohols as products are often toxic to enzymes and microorganisms, so that the amount of substrate added to the reaction system and the yield of products are very low. In addition, there is a problem that the long-term stability of the biocatalyst cannot be maintained.
【0007】したがって、上記の化学的還元法の欠点を
克服し、なおかつ既存の生物的還元法の欠陥を補うよう
な新規なカルボニル化合物の生物的還元方法の開発が強
く望まれている。Therefore, there is a strong demand for the development of a novel method for biological reduction of carbonyl compounds which overcomes the above-mentioned drawbacks of the chemical reduction method and which complements the deficiencies of the existing biological reduction methods.
【0008】[0008]
【課題を解決するための手段】そこで本発明者は、微生
物を用いるカルボニル化合物の還元方法において、基質
のカルボニル化合物や還元生成物の2級アルコール類な
どの微生物に対する毒性を回避し、水中強制分散や通気
等の面倒な操作も必要としないカルボニル化合物の微生
物的還元方法について鋭意検討した結果、栄養源および
水を含む親水性固定化担体に微生物を植菌し、これに微
生物に対して毒性を発現しない有機溶媒を接触させる
と、有機溶媒と固定化担体との界面で微生物菌体が旺盛
に増殖して担体上に菌体相を形成し、この菌体相が有機
溶媒中に添加したカルボニル化合物を高い反応速度と収
率で還元することを見出し、本発明を完成するに至っ
た。Therefore, the present inventor, in a method of reducing a carbonyl compound using a microorganism, avoids the toxicity of the carbonyl compound as a substrate or the reduction product to a microorganism such as secondary alcohols and forcibly disperses it in water. As a result of diligent study on the microbial reduction method of carbonyl compounds that does not require troublesome operations such as ventilation and aeration, the microorganisms were inoculated on a hydrophilic immobilization carrier containing a nutrient source and water, and the toxicity to the microorganisms was increased. When a non-expressing organic solvent is contacted, microbial cells vigorously grow at the interface between the organic solvent and the immobilized carrier to form a bacterial cell phase on the carrier, and this bacterial cell phase is added to the organic solvent. The inventors have found that the compound can be reduced with a high reaction rate and a high yield, and have completed the present invention.
【0009】かくして、本発明に従えば、親水性固定化
担体に、カルボニル基の還元能を有し、2級アルコール
類を生成し得る微生物を付着固定化し、該微生物の栄養
源を含む水性媒体の存在下に、カルボニル化合物を含む
実質的に水に不溶性ないしは難溶性の有機溶媒を該担体
上の固定化菌体相と接触せしめることを特徴とするカル
ボニル化合物の還元方法が提供される。[0009] Thus, according to the present invention, an aqueous medium containing a nutrient source for the microorganism, which has a carbonyl group-reducing ability and is capable of producing a secondary alcohol, is adhered and immobilized on the hydrophilic immobilization carrier. In the presence of the carbonyl compound, a substantially water-insoluble or sparingly soluble organic solvent containing the carbonyl compound is brought into contact with the immobilized bacterial cell phase on the carrier.
【0010】本発明の1つの特徴は、脂肪族炭化水素に
代表される有機溶媒と親水性固定化担体との界面で微生
物の栄養源を含む水性媒体を供給しつつ微生物を増殖さ
せて形成された菌体相を触媒として利用できる点にあ
る。これにより、カルボニル化合物を実質的に水に不溶
性もしくは難溶性の有機溶媒に溶解した形で該菌体相に
接触させて還元反応を行なわせることが可能になり、そ
の結果、高い反応速度と触媒の長期安定性が達成され、
撹拌動力が事実上不要である等の利点が得られる。One feature of the present invention is that the microorganisms are grown at the interface between an organic solvent represented by an aliphatic hydrocarbon and a hydrophilic immobilization carrier while supplying an aqueous medium containing a nutrient source for the microorganisms. This is because the bacterial cell phase can be used as a catalyst. This makes it possible to bring the carbonyl compound into contact with the bacterial cell phase in a form in which it is dissolved in an organic solvent that is substantially insoluble or sparingly soluble in water to carry out the reduction reaction, and as a result, a high reaction rate and catalyst Long-term stability of
Advantages such as the fact that stirring power is virtually unnecessary are obtained.
【0011】本発明のもう1つの特徴は、基質であるカ
ルボニル化合物は微生物に対して強い毒性を示すにもか
かわらず、これを非常に高い濃度で有機溶媒に添加する
ことができ、菌体相に接触し得る点にある。例えば、2
−オクタノンや2−デカノン等の場合には、エマルジヨ
ン系ではわずか0.5%存在するだけで微生物は増殖し
えない。しかしながら、本発明に従つて、例えばシユー
ドモナス・プチダIFO 13696は、有機溶媒中に
2−オクタノンを12%の濃度で含んでいても死滅せ
ず、また2−デカノンを24%の濃度で含んでいても死
滅せず、増殖して菌体相を形成する。本発明によれば、
このように微生物菌体相に対して高濃度のカルボニル化
合物を接触することができ、その結果、高い反応速度と
高い収率が達成され、カルボニル化合物を低コストで還
元し得る等の利点が得られる。Another feature of the present invention is that although the carbonyl compound as a substrate shows a strong toxicity to microorganisms, it can be added to an organic solvent at a very high concentration, and There is a point that can contact. For example, 2
In the case of octane and 2-decanone, microorganisms cannot grow because only 0.5% is present in the emulsion type. However, in accordance with the present invention, for example, C. petitida IFO 13696 is not killed by the inclusion of 2-octanone at a concentration of 12% in the organic solvent and 2-decanone at a concentration of 24%. Does not die, but proliferates to form a bacterial cell phase. According to the invention,
In this way, a high concentration of carbonyl compound can be contacted with the microbial cell phase, and as a result, a high reaction rate and high yield can be achieved, and advantages such as reduction of the carbonyl compound at low cost can be obtained. To be
【0012】以下、本発明についてさらに詳細に説明す
る。The present invention will be described in more detail below.
【0013】なお、上記固定化菌体相に接触させる有機
溶媒又は基質であるカルボニル化合物の有機溶媒溶液
を、以下、有機液相ということがある。The organic solvent or the organic solvent solution of the carbonyl compound, which is a substrate, which is brought into contact with the immobilized bacterial cell phase may be hereinafter referred to as an organic liquid phase.
【0014】本発明で使用可能な固定化担体は、親水性
のものであれば特に制約はなく、栄養源を含む水溶液を
含浸もしくは接触させて有機溶媒との界面に存在する微
生物にこれを供給することができるものであれば、いか
なる素材であっても使用可能であり、具体的には例え
ば、アルギン酸、カラギーナン、デンプンマトリック
ス、寒天、濾紙のようなセルロース材などの天然高分
子;ポリビニルアルコール、ウレタンポリマー、ポリア
クリルアミド、ポリアクリル酸などの合成高分子;泡ガ
ラス、シリカゲルなどの無機物などが挙げられる。The immobilization carrier usable in the present invention is not particularly limited as long as it is hydrophilic, and it is supplied to a microorganism existing at the interface with an organic solvent by impregnating or contacting an aqueous solution containing a nutrient source. Any material can be used as long as it can be used. Specifically, for example, natural polymers such as alginic acid, carrageenan, starch matrix, agar, and cellulose materials such as filter paper; polyvinyl alcohol, Examples include synthetic polymers such as urethane polymers, polyacrylamide, and polyacrylic acid; inorganic materials such as foam glass and silica gel.
【0015】これら固定化用担体の形状には特に制限は
なく、繊維状、膜状、粒状等の任意の形状に成形されて
いることができ、また布、不織布、紙、ボール紙等の形
態に成形されたものであってもよい。The shape of these immobilization carriers is not particularly limited, and they can be formed into any shape such as fibrous, film-like, and granular shapes, and can be in the form of cloth, non-woven fabric, paper, cardboard, etc. It may be molded into.
【0016】一方、上記固定化菌体相に接触する有機液
相における有機溶媒又は基質のカルボニル化合物の有機
溶媒溶液調製用の有機溶媒は、付着微生物菌体に対して
実質的に毒性を示さないものが好ましく、具体的には、
ヘキサン、ヘプタン、オクタン、ノナン、デカン等の炭
素数6〜20のメタン系炭化水素に代表されるノルマル
バラフイン類又は流動パラフイン類;イソオクタン等の
イソパラフイン類;ペンチルベンゼン、ヘキシルベンゼ
ン、ヘプチルベンゼン、オクチルベンゼン等の脂肪族鎖
の炭素数が5〜15のノルマルアルキルベンゼン類;キ
ユメン等のイソアルキルベンゼン類;シクロヘキサン等
の脂環式炭化水素類;ジエチルエーテル等のエーテル類
などを例示することができる。On the other hand, the organic solvent in the organic liquid phase in contact with the immobilized bacterial cell phase or the organic solvent for preparing the organic solvent solution of the carbonyl compound of the substrate does not substantially show toxicity to adherent microbial cells. Preferred is, specifically,
Normal paraffins or liquid paraffins represented by methane hydrocarbons having 6 to 20 carbon atoms such as hexane, heptane, octane, nonane, and decane; isoparaffins such as isooctane; pentylbenzene, hexylbenzene, heptylbenzene, Examples thereof include normal alkylbenzenes having an aliphatic chain of 5 to 15 carbon atoms such as octylbenzene; isoalkylbenzenes such as quinume; alicyclic hydrocarbons such as cyclohexane; and ethers such as diethyl ether.
【0017】上記固定化担体に付着させて、担体と有機
液体との界面で増殖させて使用するカルボニル基の還元
能を有する微生物は、細菌類、カビ類、酵母、放線菌類
等のいずれの微生物であつてもよい。具体的には例え
ば、バチルス(Bacillus)属、キサントモナス(Xantho
monas)属、ラクトバチルス(Lactobacillus)属、ロイ
コノストク(Leuconostoc)属、ストレプトコツカス(Stre
ptococcus)属、シユードモナス(Pseudomonas)属等の細
菌類、ピシア(Pichia)属、カンジダ(Candida)属、
ロドトルラ(Rhodotorula)属、サツカロマイセス(Saccha
romyces)属、シゾサツカロマイセス(Schizosacchromyce
s)属、クロエツケラ(Kloeckera)属、クリプトコツカス
(Cryptococcus)属、デイポダスカス(Dipodascus)属、
ロデロマイセス(Lodderomyces)属等の酵母類、ジオトリ
カム(Geotrichum)属、グレオスポリウム(Gloeosporiu
m)属、オーレオバシデイウム(Aureobasidium)属、アス
ペルギルス(Aspergillus)属、ペニシリウム(Penicilliu
m)属、スポロトリカム(Sporotrichum)属、クルブラリア
(Curuvlaria)属等のカビ類等に属する微生物が挙げられ
る。さらに具体的には、バチルス・スリンギエンシス
(Ba.thuringiensis)、キサントモナス・オリゼー(X
a.Oryzae)、ラクトバチルス・ブレビス(La.brevis)、
ロイコノストク・デクストラニカム(Le. dextranicu
m)、ストレプトコツカス・フエーカリス(St.faecali
s)、ピシア・テリコラ(Pi. tericola)、カンジダ・アル
ビカンス(Ca. albicans)、ロドトルラ・ルブラ(Rh. ru
bra)、サツカロマイセス・セレビシエ(Sa. cerevisia
e)、クロエツケラ・コルテイシス(Kl. corticis)、ク
リプトコツカス・マゼランス(Cr. macerans)、デイポダ
スカス・ユニヌクレアタス(Di. uninucleatus)、ロデロ
ルイセス・エロンギスポラス(Lo. elongisporus)、ジオ
トリカム・キヤンデイダム(Ge. candidum)、オーレオバ
シデイウム・プルランス(Au.pullulans)、アスペルギ
ルス・オクラセウス(As. ochraceus)、ペニシリウム・
デカムベンス(Pe. decumbens)、スポロトリカム・エキ
シル(Sp. exile)、クルブラリア・ルナタ(Cu. lunata)
等を挙げることができる。The microorganism having the ability to reduce a carbonyl group, which is attached to the immobilized carrier and is grown at the interface between the carrier and an organic liquid, can be any microorganism such as bacteria, fungi, yeasts and actinomycetes. May be Specifically, for example, Bacillus genus, Xanthonas (Xantho
monas genus, Lactobacillus genus, Leuconostoc genus, Streptococcus (Stre
Bacteria such as genus ptococcus, genus Pseudomonas, genus Pichia, genus Candida,
The genus Rhodotorula, Saccha lomyces
genus romyces, Schizosacchromyce
s), Kloeckera, Cryptococcus
(Cryptococcus) genus, Depodascus (Dipodascus) genus,
Yeasts such as the genus Loderderomyces, the genus Geotrichum, and the glioeosporiu (Gloeosporiu)
m) genus, Aureobasidium genus, Aspergillus genus, Penicilliu
genus m), genus Sporotrichum, vulbraria
(Curuvlaria) and other microorganisms belonging to molds and the like. More specifically, Bacillus thuringiensis, Xanthomonas oryzae (X.
a. Oryzae), Lactobacillus brevis,
Leuconostok Dextranicum
m), Streptococcus faecalis (St. faecali
s), Picia tericola (Pi. tericola), Candida albicans (Ca. albicans), Rhodotorula rubra (Rh. ru)
bra), Sa. cerevisia
e), Kl. corticis, Cryptococcus magerans, Cr. macerans, Di. uninucleatus, Lo. elongisporus, Ge. Aureobasidium pullulans (Au. Pullulans), Aspergillus ocraceus (As. Ochraceus), Penicillium
Decumbens, Sporotricham Exil, Cu. Lunata
Etc. can be mentioned.
【0018】かかる微生物の担体への付着固定化は、例
えば、菌体懸濁液をあらかじめ栄養源を含む水性媒体を
含ませた担体に塗布または散布するか、担体を菌体培養
液中に浸漬するか、微生物菌体を適当な方法で担体に付
着させた後、担体に栄養源を含む水性媒体を供給する等
の方法で担体上に微生物菌体を付着させた後、その担体
をあらかじめ栄養源を含む水性媒体中で培養することも
できるが、通常、基質としてのカルボニル化合物を含む
かまたは含まない有機溶媒と接触させた状態で培養し、
付着した微生物菌体を担体と有機溶媒と有機溶媒との界
面で増殖させ、担体上に固定化菌体相を形成させること
により行うのが適当できる。この培養により、微生物は
担体表面に強固に付着し、固定化菌体相が担体から剥離
するようなことはほとんどない。The adhesion and immobilization of such microorganisms on the carrier can be carried out, for example, by applying or spraying a bacterial cell suspension on a carrier containing an aqueous medium containing a nutrient source in advance, or immersing the carrier in a bacterial cell culture solution. Or, after attaching the microbial cells to the carrier by an appropriate method, after attaching the microbial cells on the carrier by a method such as supplying an aqueous medium containing a nutrient source to the carrier, the carrier is pre-nutrified. Although it can be cultured in an aqueous medium containing a source, it is usually cultured in a state of being contacted with an organic solvent containing or not containing a carbonyl compound as a substrate,
Suitably, the adhered microbial cells are grown at the interface between the carrier, the organic solvent and the organic solvent to form an immobilized microbial cell phase on the carrier. By this culturing, the microorganisms adhere strongly to the surface of the carrier, and the immobilized bacterial cell phase hardly separates from the carrier.
【0019】上記培養において使用し得る微生物の栄養
源は、使用菌体の種類に応じてその菌体に最適のものを
選択することができ、例えば、グルコース等の炭素源、
尿素等の窒素源、硫酸マグネシウム等の微量金属塩、酵
母エキス等の微量栄養源よりなる一般的なものでよい。As the nutrient source of the microorganism that can be used in the above-mentioned culture, an optimum one can be selected according to the type of the bacterial cell used, for example, a carbon source such as glucose,
A general source consisting of a nitrogen source such as urea, a trace metal salt such as magnesium sulfate, and a trace nutrient source such as yeast extract may be used.
【0020】固定化菌体への栄養源を含む水性媒体すな
わち培養液の供給は、担体が例えば寒天のように培養液
を充分に含有保持しうるものであれば、担体に予め含ま
せておくことにより行うことができ、及び/又は例え
ば、上記有機液相に培養液を加え、形成される有機液相
と培養液相の界面に微生物固定化担体を介在させること
により行なうこともできる。The aqueous medium containing the nutrient source, that is, the culture solution, is supplied to the immobilized cells in advance if the carrier can sufficiently contain and retain the culture solution, such as agar. And / or for example, by adding a culture solution to the above organic liquid phase and interposing a microorganism-immobilized carrier at the interface between the organic liquid phase and the culture liquid phase to be formed.
【0021】培養は一般に、恒温槽、インキュベーター
等の培養装置中で行うことができ、あるいは担体を基質
を含むか含まない有機溶媒中に浸漬し、場合によっては
さらに栄養源を含む水性媒体を加えた反応容器中で温度
調節しながら行ってもよい。培養温度、培養時間等の培
養条件は使用微生物の種類に応じて、最適の条件を選択
することができる。Culturing can generally be carried out in a culturing device such as a thermostat or an incubator, or the carrier is immersed in an organic solvent containing or not containing a substrate, and optionally an aqueous medium containing a nutrient source is added. It may be carried out while controlling the temperature in the reaction vessel. The optimal culture conditions such as culture temperature and culture time can be selected according to the type of microorganism used.
【0022】培養に酸素を必要とする場合には、有機溶
媒に通気すればよいが、一般に有機溶媒は水に比して数
倍から十数倍の酸素溶解度を有しているため、かならず
しも通気する必要はない。また、酸素が培養または反応
の妨げになる場合には、有機溶媒中の溶存酸素を二酸化
炭素、窒素等により置換すればよい。これら置換気体の
有機溶媒中への溶解性も一般に水の数倍高いため効率的
に置換が行える。一方、培養中の撹拌についても、基質
であるカルボニル化合物は有機溶媒中に溶解して存在し
ているため、エマルジヨン法のような強烈な撹拌は不要
でありまた、撹拌が不要である場合が多い。When oxygen is required for culturing, it may be aerated with an organic solvent, but in general, an organic solvent has oxygen solubility of several times to several tens of times that of water, so that it is always aerated. do not have to. When oxygen interferes with the culture or reaction, the dissolved oxygen in the organic solvent may be replaced with carbon dioxide, nitrogen, or the like. Since the solubility of these substitution gases in the organic solvent is generally several times higher than that of water, substitution can be performed efficiently. On the other hand, as for the stirring during culture, the carbonyl compound that is the substrate is dissolved and present in the organic solvent, so that vigorous stirring such as the emulsion method is not necessary, and the stirring is often unnecessary. .
【0023】基質としてのカルボニル化合物は、上記培
養の初期から添加してもよく、または微生物が十分に増
殖して固定化菌体相を形成した後に添加してもよい。あ
るいは培養初期から固定化菌体相形成までの任意の時点
で加えてもよい。上記のように、カルボニル化合物は微
生物に対して毒性を発現する場合が多いため、一般に
は、菌体相が十分に成長してから添加する方が高い成績
が達成される。The carbonyl compound as a substrate may be added from the initial stage of the above culture, or may be added after the microorganism has sufficiently grown to form an immobilized bacterial cell phase. Alternatively, it may be added at any time from the initial stage of culture to the formation of the immobilized bacterial cell phase. As described above, since carbonyl compounds often exhibit toxicity to microorganisms, generally, higher results are achieved by adding them after the bacterial cell phase has sufficiently grown.
【0024】かくして、担体上の固定化菌体相を、基質
としてのカルボニル化合物の有機溶媒溶液からなる有機
液相との接触状態で培養をつづけることにより、カルボ
ニル化合物の還元反応を行なわせることができる。Thus, the reduction reaction of the carbonyl compound can be carried out by continuing the culture of the immobilized bacterial cell phase on the carrier in contact with the organic liquid phase consisting of the organic solvent solution of the carbonyl compound as the substrate. it can.
【0025】この固定化微生物によるカルボニル化合物
の還元反応に基質として供しうるカルボルニル化合物は
特に制限されず、固定化微生物のカルボニル基の還元能
に応じて各種のものを使用することができる。The carbonyl compound that can be used as a substrate for the reduction reaction of the carbonyl compound by the immobilized microorganism is not particularly limited, and various compounds can be used depending on the reducing ability of the carbonyl group of the immobilized microorganism.
【0026】基質として供しうるカルボニル化合物とし
ては、例えば、アセトン、メチルエチルケトン、メチル
n−プロピルケトン、メチルイソプロピルケトン、メチ
ルイソブチルケトン、ジエチルケトン、2−ヘキサノ
ン、3−ヘキサノン、2−オクタノン、2−デカノン等
のアルカノン類;3−ケトブタン酸エチル、4−クロロ
−3−ケトブタン酸エチル等のケトエステル類;アセチ
ルアセトン、アセトニルアセトン、2,4−オクタンジ
オン等のジケトン類;アセトフエノン、ホセンゾフエノ
ン、ベンジル、2−アセトキシ−1−フエニルエタノン
等の芳香族カルボニル化合物;シクロヘキサノン、2−
メチルシクロヘキサノン等の脂環式カルボニル化合物等
が挙げられる。Examples of the carbonyl compound that can serve as a substrate include acetone, methyl ethyl ketone, methyl n-propyl ketone, methyl isopropyl ketone, methyl isobutyl ketone, diethyl ketone, 2-hexanone, 3-hexanone, 2-octanone, 2-decanone. Alkanones such as; ketoesters such as ethyl 3-ketobutanoate, ethyl 4-chloro-3-ketobutanoate; diketones such as acetylacetone, acetonylacetone, 2,4-octanedione; acetophenone, fosenzophenone, benzyl, 2- Aromatic carbonyl compounds such as acetoxy-1-phenylethanone; cyclohexanone, 2-
Examples thereof include alicyclic carbonyl compounds such as methylcyclohexanone.
【0027】有機溶媒中のカルボニル化合物の濃度は特
に制限されるものではなく、菌体に対する毒性に応じて
決めることができる。カルボニル化合物が実質的に水不
溶性ないしは難溶性の場合には、これらが水相すなわち
親水性固定化担体側へ移行しないため、その菌体に対す
る毒性を大幅に減じることができる。例えば、2−オク
タノンや2−デカノンのような中鎖脂肪族カルボニル化
合物の場合には、サツカロマイセス属、ロデロマイセス
属、ピシア属やカンジダ属等の酵母は、有機液相中の濃
度が3〜5%であつても増殖可能である。一方、アセト
ン、メチルエチルケトン等のような水溶性のカルボニル
化合物の場合には、これらが水相側に移行して菌体に対
して毒性を発現する場合があるので、菌株の耐性に応じ
て濃度を決める必要がある。The concentration of the carbonyl compound in the organic solvent is not particularly limited and can be determined depending on the toxicity to the microbial cells. When the carbonyl compound is substantially water-insoluble or sparingly soluble, it does not migrate to the water phase, that is, the hydrophilic immobilization carrier side, and thus the toxicity to the bacterial cells can be greatly reduced. For example, in the case of a medium-chain aliphatic carbonyl compound such as 2-octanone or 2-decanone, yeasts such as Satsucaromyces, Roderomyces, Picia and Candida have a concentration in the organic liquid phase of 3 to 5%. However, it can grow. On the other hand, in the case of water-soluble carbonyl compounds such as acetone and methyl ethyl ketone, these may migrate to the water phase side and develop toxicity to the bacterial cells, so the concentration should be adjusted according to the resistance of the strain. I need to decide.
【0028】以上述べた本発明の方法によれば、脂肪
族、芳香族、脂環式等のカルボニル化合物の還元反応を
固定化微生物の増殖菌体を用いて、極めて効率的に行な
うことができる。その際、副反応が生ずる可能性がある
場合には、適当な代謝又は変換阻害剤の添加によつてそ
れを遮断するか又はそのような副反応が生じないように
育種改良した代謝欠損株を用いることができる。According to the above-mentioned method of the present invention, the reduction reaction of an aliphatic, aromatic, alicyclic carbonyl compound, etc. can be carried out extremely efficiently by using the proliferating cells of the immobilized microorganism. . At that time, if a side reaction is likely to occur, a metabolic deficient strain that has been bred and improved by blocking it by the addition of an appropriate metabolism or conversion inhibitor or preventing such side reaction from occurring is selected. Can be used.
【0029】本発明の方法によれば、基質のカルボニル
化合物は有機液相側から供給され、生産物のカルボニル
基還元化合物すなわち2級アルコール類は有機液相側に
蓄積される。従つて、有機液相に蓄積される生産物を回
収し、基質のカルボニル化合物を補充する等の方法を行
なうことにより、固定化菌体相と基質との接触頻度を飛
躍的に増加せしめることができ、反応速度と収率、収量
を大幅に向上させることが可能となり、連続操業も可能
となる。According to the method of the present invention, the carbonyl compound of the substrate is supplied from the organic liquid phase side, and the carbonyl group-reducing compound of the product, that is, the secondary alcohol is accumulated on the organic liquid phase side. Therefore, the frequency of contact between the immobilized cell phase and the substrate can be dramatically increased by collecting the product accumulated in the organic liquid phase and supplementing the substrate with a carbonyl compound. The reaction rate, yield, and yield can be significantly improved, and continuous operation is also possible.
【0030】かくして、本発明の方法を、例えば工業薬
品、医薬品、化粧品、香料、洗剤、界面活性剤、繊維処
理剤、油脂、染料、塗料、印刷材料等の分野における工
業上重要な2級アルコール類の製造工程に適用すること
により、生産コストの低下、工程の省エネルギー化、省
力化等、工業的に有利な種々の利点を得ることができ
る。Thus, the method of the present invention can be applied to industrially important secondary alcohols in the fields of, for example, industrial chemicals, pharmaceuticals, cosmetics, fragrances, detergents, surfactants, fiber treating agents, oils and fats, dyes, paints and printing materials. By applying it to manufacturing processes of the same class, it is possible to obtain various industrially advantageous advantages such as reduction of production cost, energy saving and labor saving of processes.
【0031】[0031]
【実施例】以下、本発明を実施例によりさらに具体的に
説明するが、本発明はこれらの実施例に限定されるもの
ではない。なお、部及び%は重量基準である。EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples. Parts and% are based on weight.
【0032】実施例1 ペプトン0.5%、酵母エキス0.3%、麦芽エキス0.
3%、グルコース1.0%、寒天1.5%よりなる寒天平
板をガラスシャーレ(表面積38.5cm2)に調製し、表
1にある各種微生物の懸濁液100μlをコンラージ棒
を用いて塗沫した。乾燥後、10%2−デカノンのn−
ヘキサデカン溶液を10ml重層し、30℃で7日間静
置培養した。培養後、溶媒相1μlをガスクロマトグラ
フィーによって分析し、生成した2オクチル酸n−ブチ
ルエステル濃度を測定した。その結果を表1に示すExample 1 Peptone 0.5%, yeast extract 0.3%, malt extract 0.1%
An agar plate consisting of 3%, glucose 1.0% and agar 1.5% was prepared in a glass petri dish (surface area 38.5 cm 2 ), and 100 μl of suspensions of various microorganisms shown in Table 1 were coated with a conradi stick. It spattered. After drying, 10% 2-decanone n-
Hexadecane solution (10 ml) was overlaid and statically cultured at 30 ° C. for 7 days. After culturing, 1 μl of the solvent phase was analyzed by gas chromatography to measure the concentration of the produced 2-octyl acid n-butyl ester. The results are shown in Table 1.
【0033】[0033]
【表1】 実施例2 実施例1と同様の培地100mlを内容量480mlの
密栓可能なガラス容器に注いで寒天平板を調製した(表
面積43cm2)。これに表2に示す各種微生物懸濁液1
00μlをコンラージ棒を用いて塗沫し、30℃で2日
間培養して菌体相を形成させた。その後、10% 2−
オクタンのn−ヘキサデカン溶液を10ml重層し、30
℃、100rpmの振盪下で7日間培養した。培養後、溶
媒相1μlをガスクロマトグラフィーによつて分析し、
生成した2−オクタノール濃度を測定した。その結果を
表2に示す。[Table 1] Example 2 100 ml of the same medium as in Example 1 was poured into a glass container having an inner volume of 480 ml which can be sealed, to prepare an agar plate (surface area 43 cm 2 ). Various microbial suspensions 1 shown in Table 2
00 μl was smeared with a conradi stick and cultured at 30 ° C. for 2 days to form a bacterial cell phase. Then 10% 2-
Overlay 10 ml of n-hexadecane solution of octane,
Culturing was carried out for 7 days under shaking at 100 ° C at 100 ° C. After culturing, 1 μl of the solvent phase was analyzed by gas chromatography,
The produced 2-octanol concentration was measured. The results are shown in Table 2.
【0034】[0034]
【表2】 実施例3 厚さ3mmの濾過板(東洋アドバンテツク製)を縦にして充
填したガラス容器(内容量500ml)を調製した(表面
積800cm2)。これに、ペプトン0.5%、酵母エキス
0.3%、麦芽エキス0.3%、グルコース1.0%、ス
パン−80 0.02%よりなる液体培地200mlを注
入し、高圧蒸気滅菌した。冷却後、これにロデロマイセ
ス・エロンギスポラスの1日培養液50mlを注入し、
0.2vvmの通気、300rpmの撹拌下で2日間培養し
た。培養後、培養液を抜きながら10%2−デカノンの
n−パラフイン溶液を注入し、培養液50ml、溶媒相1
50mlとして、30℃、300rpmの撹拌下、無通気で
7日間培養した。培養期間中、溶媒相1μlをガスクロ
マトグラフイーにより分析し、生成2−デカノール濃度
を測定した。その結果を表3に示す。[Table 2] Example 3 A glass container (internal volume: 500 ml) vertically packed with a 3 mm-thick filter plate (manufactured by Toyo Advantech) was prepared (surface area: 800 cm 2 ). To this, 200 ml of a liquid medium consisting of 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, 1.0% glucose and 0.080% span-80 was added and sterilized under high pressure steam. After cooling, inject 50 ml of Roderomyces elongisporus daily culture solution into it,
The cells were cultured for 2 days under aeration of 0.2 vvm and stirring at 300 rpm. After culturing, 10% 2-decanone n-paraffin solution was injected while removing the culture solution, and 50 ml of the culture solution, solvent phase 1
50 ml was cultured at 30 ° C. under stirring at 300 rpm for 7 days without aeration. During the culture period, 1 μl of the solvent phase was analyzed by gas chromatography to measure the concentration of 2-decanol produced. The results are shown in Table 3.
【0035】[0035]
【表3】 実施例4 ポリペプトン1.0%、酵母エキス0.2%、硫酸マグネ
シウム0.1%、寒天1.5%よりなる寒天平板をガラス
シャーレに調製し(表面積38.5cm2)、これにラクト
バチルス・ブレビスIAM 1082の1日培養液10
0μlをコンラージ棒を用いて植菌し、溶存酸素を窒素
で置換したn−パラフイン10mlを重層した。30℃で
2日間静置培養して菌体相を形成された後4−メチルシ
クロヘキサノンを200μl添加溶解し、7日間静置培
養した。培養後、溶媒相から1μlを採取し、ガスクロ
マトグラフィーを用いて生成2−メチルシクロヘキサノ
ール濃度を測定した。その結果、1.2gの2−メチル
シクロヘキサノールの蓄積を確認した。[Table 3] Example 4 An agar plate consisting of 1.0% polypeptone, 0.2% yeast extract, 0.1% magnesium sulfate and 1.5% agar was prepared in a glass petri dish (surface area 38.5 cm 2 ), and Lactobacillus. Brevis IAM 1082 daily culture solution 10
0 μl was inoculated with a conradi rod and 10 ml of n-paraffin in which dissolved oxygen was replaced with nitrogen was overlaid. After stationary culture at 30 ° C. for 2 days to form a bacterial cell phase, 200 μl of 4-methylcyclohexanone was added and dissolved, and stationary culture was carried out for 7 days. After the culturing, 1 μl was collected from the solvent phase, and the concentration of produced 2-methylcyclohexanol was measured using gas chromatography. As a result, accumulation of 1.2 g of 2-methylcyclohexanol was confirmed.
【0036】比較例1 ペプトン0.5%、酵母エキス0.3%、麦芽エキス0.
3%、グルコース1.0%、スパン−80 0.02%よ
りなる液体培地にロデロマイセス・エロンギスポラスを
植菌して1日間30℃で振盪培養した。これに2−オク
タノンを0.2、0.4、0.6、0.8、1.0%になるよ
うに添加し7日間振盪培養した。培養後、ジエチルエー
テルで3回抽出し、乾燥、希釈後、ガスクロマトグラフ
イーにより、生成2−オクタノール濃度を測定した。そ
の結果、いずれの濃度においても、生成2−オクタノー
ル濃度は0.1g/l以下であつた。Comparative Example 1 Peptone 0.5%, yeast extract 0.3%, malt extract 0.1%.
A liquid medium consisting of 3%, glucose 1.0%, and span-80 0.02% was inoculated with Roderomyces elongisporus, and cultured at 30 ° C. for 1 day with shaking. 2-Octanone was added to this so as to be 0.2, 0.4, 0.6, 0.8, and 1.0%, and shake culture was carried out for 7 days. After culturing, the mixture was extracted three times with diethyl ether, dried and diluted, and the concentration of produced 2-octanol was measured by gas chromatography. As a result, the produced 2-octanol concentration was 0.1 g / l or less at any concentration.
【0037】比較例2 寒天を除き、スパン−80を0.02%添加して窒素置
換した実施例1の培地20mlにラクトバチルス・ブレビ
スIAM 1082の1日培養液を1ml添加し、1日間
増殖させた。その後、2−メチルシクロヘキサノンを
0.2、0.4、0.6、0.8、1.0%になるように培
地中に添加し、7日間150rpmで振盪培養した。培養
後、ジエチルエーテルで3回抽出し、乾燥、希釈後、ガ
スクロマトグラフイーを用いて生成2−メチルシクロヘ
キサノール濃度を測定した。その結果、いずれの添加濃
度においても、生成2−メチルシクロヘキサノール濃度
は0.1g/l以下であつた。Comparative Example 2 1 ml of a 1-day culture of Lactobacillus brevis IAM 1082 was added to 20 ml of the medium of Example 1 which had been replaced with nitrogen by adding 0.02% of Span-80 and nitrogen substitution, and grown for 1 day. Let Then, 2-methylcyclohexanone was added to the medium so that the concentration was 0.2, 0.4, 0.6, 0.8, and 1.0%, and the mixture was cultured with shaking at 150 rpm for 7 days. After culturing, the mixture was extracted three times with diethyl ether, dried and diluted, and the concentration of produced 2-methylcyclohexanol was measured using gas chromatography. As a result, the produced 2-methylcyclohexanol concentration was 0.1 g / l or less at any addition concentration.
───────────────────────────────────────────────────── 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 (C12P 7/02 C12R 1:84) (C12P 7/02 C12R 1:645) (C12P 7/02 C12R 1:78) ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Internal reference number FI Technical display location (C12P 7/02 C12R 1:84) (C12P 7/02 C12R 1: 645) (C12P 7/02 C12R 1:78)
Claims (1)
元能を有し2級アルコール類を生成し得る微生物を付着
固定化し、該微生物の栄養源を含む水性媒体の存在下
に、カルボニル化合物を含む実質的に水に不溶性ないし
は難溶性の有機溶媒を該担体上の固定化菌体相と接触せ
しめることを特徴とするカルボニル化合物の還元方法。1. A carbonyl compound in the presence of an aqueous medium containing a nutrient source for said microorganism, wherein a microorganism capable of reducing a carbonyl group and capable of producing a secondary alcohol is adhered and immobilized on a hydrophilic immobilization carrier. A method for reducing a carbonyl compound, which comprises contacting a substantially water-insoluble or sparingly water-soluble organic solvent containing the above with the immobilized bacterial cell phase on the carrier.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP18464392A JPH0688A (en) | 1992-06-19 | 1992-06-19 | Method for reducing carbonyl compound with immobilized microorganism |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP18464392A JPH0688A (en) | 1992-06-19 | 1992-06-19 | Method for reducing carbonyl compound with immobilized microorganism |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH0688A true JPH0688A (en) | 1994-01-11 |
Family
ID=16156826
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP18464392A Pending JPH0688A (en) | 1992-06-19 | 1992-06-19 | Method for reducing carbonyl compound with immobilized microorganism |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH0688A (en) |
-
1992
- 1992-06-19 JP JP18464392A patent/JPH0688A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
FI68078B (en) | FOERFARANDE FOER FRAMSTAELLNING AV BIOLOGISKT AKTIVA MICROORGANISMMYCELIEPELLETS | |
Bezbradica et al. | Immobilization of yeast cells in PVA particles for beer fermentation | |
McGhee et al. | Ethanol production by immobilized Saccharomyces cerevisiae, Saccharomyces uvarum, and Zymomonas mobilis | |
Ogawa et al. | Production of kojic acid by membrane-surface liquid culture of Aspergillus oryzae NRRL484 | |
EP0789079B1 (en) | Coupling process of fermentation and microbial transformation reaction | |
Halan et al. | Catalytic Pseudomonas taiwanensis VLB120ΔC biofilms thrive in a continuous pure styrene generated by multiphasic segmented flow in a capillary microreactor | |
EP0756002B1 (en) | Interface bioreactor system | |
Pakula et al. | A new continuous biofilm bioreactor for immobilized oil‐degrading filamentous fungi | |
JPH0688A (en) | Method for reducing carbonyl compound with immobilized microorganism | |
Arica et al. | Covalent immobilization of Aspergillus niger on pHEMA membrane: application to continuous flow reactors | |
JPH0690A (en) | Method for oxidizing alcohols with immobilized microorganism | |
Darah et al. | Laboratory-scale production of lignin-degrading enzymes by free and entrapped cells of Phanerochoete chrysosporium in a tubular air-lift bioreactor | |
Murdin et al. | Growth and metabolism of hybridomas immobilized in packed beds: comparison with static and suspension cultures | |
JP2542766B2 (en) | Immobilization method of microorganisms and non-aqueous microbial conversion reaction method by immobilized microorganisms | |
Gbewonyo et al. | [28] Immobilization of mycelial cells on celite | |
Webb et al. | The role of chemical engineering in biotechnology | |
JPH11225740A (en) | High layer type interfacial bioreactor | |
JPH0630594B2 (en) | Method for industrial production of polyol by fermentation of sugars | |
JPH06197777A (en) | Transesterification by immobilized microorganism | |
JPH06197773A (en) | Hydrolysis of esters with immobilized microorganism | |
EP1114869A1 (en) | Process for producing ursodeoxycholate | |
JP4268719B2 (en) | Anaerobic flat plate interface bioreactor | |
JPH04316489A (en) | Production of optically active (s)-3-chloro-1-phenyl-1-propanol | |
JP2000300289A (en) | Production of (s)-ibuprofen | |
JPH09131198A (en) | Fusion of fermentation with microbial conversion reaction |