JPS638759B2 - - Google Patents
Info
- Publication number
- JPS638759B2 JPS638759B2 JP18950181A JP18950181A JPS638759B2 JP S638759 B2 JPS638759 B2 JP S638759B2 JP 18950181 A JP18950181 A JP 18950181A JP 18950181 A JP18950181 A JP 18950181A JP S638759 B2 JPS638759 B2 JP S638759B2
- Authority
- JP
- Japan
- Prior art keywords
- coenzyme
- pore volume
- silica gel
- crude
- chromatography
- 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.)
- Expired
Links
- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 claims description 53
- 235000017471 coenzyme Q10 Nutrition 0.000 claims description 41
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 31
- 239000011148 porous material Substances 0.000 claims description 27
- 239000000741 silica gel Substances 0.000 claims description 19
- 229910002027 silica gel Inorganic materials 0.000 claims description 19
- 239000000499 gel Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 6
- 239000000377 silicon dioxide Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 5
- 238000010898 silica gel chromatography Methods 0.000 claims description 5
- 239000012046 mixed solvent Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 3
- 150000001338 aliphatic hydrocarbons Chemical class 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims 2
- 238000012856 packing Methods 0.000 claims 1
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 11
- 238000005194 fractionation Methods 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 238000004816 paper chromatography Methods 0.000 description 3
- 241000222178 Candida tropicalis Species 0.000 description 2
- 241000223254 Rhodotorula mucilaginosa Species 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000000704 physical effect Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000010977 unit operation Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 1
- 206010007559 Cardiac failure congestive Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 238000012369 In process control Methods 0.000 description 1
- 241000288902 Lemur catta Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- -1 aliphatic alcohols Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000027721 electron transport chain Effects 0.000 description 1
- 239000012156 elution solvent Substances 0.000 description 1
- GCFHZZWXZLABBL-UHFFFAOYSA-N ethanol;hexane Chemical compound CCO.CCCCCC GCFHZZWXZLABBL-UHFFFAOYSA-N 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- ZKQFHRVKCYFVCN-UHFFFAOYSA-N ethoxyethane;hexane Chemical compound CCOCC.CCCCCC ZKQFHRVKCYFVCN-UHFFFAOYSA-N 0.000 description 1
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000010965 in-process control Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 239000012454 non-polar solvent Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 238000004886 process control Methods 0.000 description 1
- 150000004059 quinone derivatives Chemical class 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Description
本発明は、補酵素Ｑの製造方法に係り、更に詳
しくは微生物菌体等から得られた粗製補酵素Ｑを
高収率、高純度で回収する精製補酵素Ｑの製造方
法に関するものである。
補酵素Ｑは微生物・動物の細胞中のミトコンド
リアに主として存在し末端電子伝達系に関与する
重要なキノン誘導体で、近年医薬としての特性が
注目され、うつ血性心不全の治療薬として用いら
れているほか多くの薬理的効果が見い出されつつ
あり、今後その医薬品としての用途がさらに拡大
されることが期待されているものである。
従来の一般的な補酵素Ｑの精製法として用いら
れてきたシリカゲルクロマトグラフイーでは、工
業的規模での大量処理を行なおうとすれば分画能
が低下するため収率が低く、さらに精製工程を設
けなければ高純度品が得られず、その上使用する
ゲルの微妙な品質変動により分画能が左右され安
定した精製効果を得る為の工程管理は容易ではな
かつた。
本発明は、かかる従来法の欠点を除き工業的に
有利なカラムクロマトグラフイー手段を確立する
ため研究した結果完成されたものである。
通常、微生物菌体等から抽出して得られた粗製
補酵素Ｑ含有液を市販のシリカゲルに吸着させた
後、極性溶媒あるいは非極性溶媒を適宜に組み合
わせて使用する等により補酵素Ｑを分画精製す
る。この場合、従来のクロマトグラフイーにおい
ては、シリカゲルの品種−溶媒系の組み合わせの
選定がその分画性能を支配する最大のポイントで
あるにもかかわらず、その選定は経験的に行なわ
れることが多かつた。また大量に使用する場合に
はロツトごとにゲルの物性が変動することは避け
られず、それに伴なう分画能の変動は工程管理上
大きな問題であつた。また分画能の変動は負荷試
料成分組成の変動あるいは洗浄、溶出溶剤の組成
変動によつても引き起されるので、その解析は容
易ではなかつた。
本発明者らは工程管理上のポイントをゲル物性
に求め、種々研究を重ねた結果、シリカゲルの表
面積によつて分画性能に相違があり、負荷試料成
分組成や展開溶媒の種類など、他の条件が一定の
もとでは、最適の表面積値があることを発見し
た。従つて、市販のシリカゲルをそのまま使用せ
ず、１操作に使用するゲルの表面積を特定の値に
調整して使用することが大規模処理において安定
な分画能を得る為に極めて有効であることを見い
出した。しかしシリカゲルの表面積を測定するの
は煩雑である為、その代用特性として細孔容積を
使用すると便利である。細孔容積と表面積との間
には、図１に示す関係にあることが知られてい
る。
細孔容積は、一定量のゲルに純水を滴下しゲル
の流動性がなくなり、塑性が出るポイントを終点
としたとき、滴下した純水の量により求めること
ができる。
微生物菌体から抽出された精製補酵素Ｑ含有液
を、そのままあるいは前処理、濃縮後、細孔容積
を前記方法により測定した、異なる細孔容積値を
もつ複数種のシリカゲルを特定の細孔容積になる
ように混合した後、カラムに充填し該粗製補酵素
Ｑ含有液を負荷して適当な組成および濃度の溶媒
系で溶出し補酵素Ｑ画分を分画精製する。得られ
た補酵素Ｑ画分からはアセトン、メタノール、エ
タノール等で結晶化を行なつて一層高純度の補酵
素Ｑを製造することができる。
採用するこの細孔容積の特定値は、精製の条
件、たとえば粗製補酵素Ｑ含有液の性質（補酵素
Ｑの種類や不純物にもとづく）や、使用する溶媒
系の種類等によつて異なる。しかし、いずれのケ
ースでも各々の条件下で特定の細孔容積値を選
び、シリカゲルカラム処理のとき使用するシリカ
ゲルの細孔容積値を常にその特定値に設定して操
作することにより高い純度と回収率を安定維持す
る事が出来る。特定値の選び方については、例え
ば異なる細孔容積値をもつシリカゲルを混合して
各種の細孔容積値をもつシリカゲルを調製し、シ
リカゲルカラムクロマトグラフイーの単位操作に
おける高純度と高回収率を判断基準にして決定す
るのが普通であるが、これに限られるものではな
い。例えば、後の再結晶工程の収率純度、使用す
る溶媒の扱い易さなどを考慮に入れ、シリカゲル
カラムの単位操作だけでなく、全収率、最終純
度、作業性等を考慮して選ぶこともできる。
ここで選ばれる細孔容積の特定値は、前記の目
的にかなう値なればよく特に制限はないが、通常
0.70〜1.10ml／ｇ、さらに0.80〜0.90ml／ｇの範
囲の値であることが多い。
粗製補酵素Ｑ含有液は、動物、植物あるいは微
生物などの起源、およびその後本発明を適用する
に到るまでの処理の過程を問わない。又、目的と
する補酵素Ｑの種類も特に制限はないが、Q6、
Q7、Q8、Q9あるいはQ10に適用した場合、所期の
効果が顕著である。さらにシリカゲルクロマトに
使用する展開溶媒も通常補酵素Ｑに使用されるも
のならばよく、とくに制限はないが、前記の補酵
素Ｑ類については、C5〜C8の炭化水素、とくに
ｎ−ヘキサン、ｎ−ヘプタン等の脂肪族炭化水
素、エチルエーテル等エーテル系、酢酸エチル、
酢酸ブチル等エステル系、メチルエチルケトンな
どケトン系の有機溶媒、もしくはこれらを組合わ
せた混合溶媒がよく使用される。とくに、ｎ−ヘ
キサンとエチルエーテル、ｎ−ヘキサンと酢酸エ
チルの混合溶媒は本発明の目的に沿う場合が多
い。又、C3〜C6の脂肪族アルコールも適用でき
る。展開もしくは溶出の方法は、通常クロマトグ
ラフイーに使用される方法に対して通用でき、特
に制限はない。
以下実施例によつて具体的に例示するが、本発
明はこれらの例にのみ限定されるものではない。
実施例 １
補酵素Q10を生産するロードトルラ・ムシラギ
ノーザ（Rhodotorula mucilaginosa）AHU−
3946の培養液40から菌体を分離し、酸処理、ア
ルカリ処理を行つた後、エタノール−ｎ−ヘキサ
ン混合物（１：４）を８添加し60℃で２時間抽
出し、ヘキサン層を濃縮乾固して補酵素Q10を含
有する油状物質19.24ｇ（CoQ10として4.80ｇ）を
得た。これを四等分してｎ−ヘキサンに溶解し以
下の操作を行つた。
メルク社製シリカゲル60（細孔容積0.75ml／ｇ、
表面積500m2／ｇ）およびシリカゲル100（細孔容
積1.00ml／ｇ、表面積400m2／ｇ）を混合調整し
たシリカゲル24ｇを充填したカラムに負荷し1.0
％酢酸エチル−ｎ−ヘキサンにて洗浄、溶出を行
いCoQ10画分を得た。これらをさらにエタノール
にて晶析を行い、得られた試料の純度測定を行つ
た。以上の結果をまとめると表１のようになる。
試料はマススペクトル、逆相ペーパークロマトグ
ラフイーにより補酵素Q10であると同定された。
The present invention relates to a method for producing coenzyme Q, and more particularly to a method for producing purified coenzyme Q, in which crude coenzyme Q obtained from microbial cells is recovered with high yield and purity. Coenzyme Q is an important quinone derivative that mainly exists in the mitochondria of microbial and animal cells and is involved in the terminal electron transport chain.In recent years, its medicinal properties have attracted attention, and it is used as a treatment for congestive heart failure. Many pharmacological effects are being discovered, and it is expected that its use as a drug will further expand in the future. Silica gel chromatography, which has been conventionally used as a general purification method for coenzyme Q, has a low yield due to a decrease in fractionation ability when large-scale processing is attempted on an industrial scale. High purity products cannot be obtained without the provision of a filter, and on top of that, the fractionation ability is affected by slight variations in the quality of the gel used, making it difficult to control the process to obtain a stable purification effect. The present invention was completed as a result of research aimed at establishing an industrially advantageous column chromatography method that eliminates the drawbacks of the conventional methods. Usually, a crude coenzyme Q-containing solution obtained by extraction from microbial cells is adsorbed onto commercially available silica gel, and then coenzyme Q is fractionated by using an appropriate combination of polar or non-polar solvents. refine. In this case, in conventional chromatography, the selection of the combination of silica gel type and solvent system is the most important point that governs its fractionation performance, but this selection is often made empirically. Katta. Furthermore, when using in large quantities, it is inevitable that the physical properties of the gel will vary from lot to lot, and the resulting variation in fractionation ability has been a major problem in terms of process control. In addition, variations in fractionation ability are caused by variations in the composition of loaded sample components, or variations in the composition of washing and elution solvents, so analysis thereof has not been easy. The present inventors looked to the physical properties of the gel as a key point in process control, and as a result of various studies, it was found that the fractionation performance differs depending on the surface area of the silica gel. It was discovered that under certain conditions there is an optimum surface area value. Therefore, rather than using commercially available silica gel as is, it is extremely effective to adjust the surface area of the gel used in one operation to a specific value in order to obtain stable fractionation performance in large-scale processing. I found out. However, since it is complicated to measure the surface area of silica gel, it is convenient to use pore volume as a substitute characteristic. It is known that there is a relationship between pore volume and surface area as shown in FIG. The pore volume can be determined by the amount of pure water dropped into a certain amount of gel when the end point is the point at which the gel loses its fluidity and becomes plastic. The pore volume of a purified coenzyme Q-containing solution extracted from microbial cells was measured by the method described above, either as is or after pretreatment and concentration. After mixing the column, the crude coenzyme Q-containing solution is loaded into a column and eluted with a solvent system having an appropriate composition and concentration to fractionate and purify the coenzyme Q fraction. The obtained coenzyme Q fraction can be crystallized with acetone, methanol, ethanol, etc. to produce even higher purity coenzyme Q. The specific value of this pore volume to be adopted varies depending on the purification conditions, for example, the properties of the crude coenzyme Q-containing solution (based on the type of coenzyme Q and impurities), the type of solvent system used, etc. However, in each case, high purity and recovery can be achieved by selecting a specific pore volume value under each condition and always setting the pore volume value of the silica gel used during silica gel column treatment to that specific value. It is possible to maintain a stable rate. Regarding how to select a specific value, for example, mix silica gels with different pore volume values to prepare silica gels with various pore volume values, and judge high purity and high recovery rate in unit operation of silica gel column chromatography. Usually, it is determined based on a standard, but it is not limited to this. For example, take into account the yield purity of the subsequent recrystallization step, the ease of handling the solvent used, etc., and select it by considering not only the unit operation of the silica gel column, but also the overall yield, final purity, workability, etc. You can also do it. The specific value of the pore volume selected here is not particularly limited as long as it satisfies the above purpose, but usually
Values often range from 0.70 to 1.10 ml/g, and even from 0.80 to 0.90 ml/g. The crude coenzyme Q-containing solution may be of any animal, plant or microorganism origin, and regardless of the treatment process used until the present invention is applied. In addition, there is no particular restriction on the type of coenzyme Q to be used, but Q 6 ,
The intended effect is significant when applied to Q 7 , Q 8 , Q 9 or Q 10 . Furthermore, the developing solvent used in silica gel chromatography may be one normally used for coenzyme Q, and is not particularly limited . , aliphatic hydrocarbons such as n-heptane, ethers such as ethyl ether, ethyl acetate,
Ester-based organic solvents such as butyl acetate, ketone-based organic solvents such as methyl ethyl ketone, or a mixed solvent of these are often used. In particular, mixed solvents of n-hexane and ethyl ether and n-hexane and ethyl acetate often meet the objectives of the present invention. Furthermore, C3 to C6 aliphatic alcohols can also be applied. The development or elution method can be applied to methods normally used in chromatography and is not particularly limited. The present invention will be specifically illustrated by Examples below, but the present invention is not limited only to these Examples. Example 1 Rhodotorula mucilaginosa AHU- producing coenzyme Q 10
After separating the bacterial cells from 40°C of culture solution of 3946 and performing acid treatment and alkali treatment, 88% of ethanol-n-hexane mixture (1:4) was added and extracted at 60°C for 2 hours, and the hexane layer was concentrated and dried. After solidification, 19.24 g of an oily substance containing coenzyme Q 10 (4.80 g as CoQ 10 ) was obtained. This was divided into four equal parts, dissolved in n-hexane, and the following operations were performed. Silica gel 60 manufactured by Merck (pore volume 0.75ml/g,
A column packed with 24 g of silica gel prepared by mixing and adjusting silica gel 100 (pore volume 1.00 ml/g, surface area 400 m 2 /g) was loaded with 1.0
Washing and elution were performed with % ethyl acetate-n-hexane to obtain 10 CoQ fractions. These were further crystallized with ethanol, and the purity of the obtained sample was measured. Table 1 summarizes the above results.
The sample was identified as coenzyme Q 10 by mass spectrometry and reversed phase paper chromatography.
【表】
表１のデータとは別に、補酵素Q10を生産する
ロードトルラ・ムシラギノーザAHU3946を、表
１の場合と同じ条件で20培養し、同様の処理を
行つて補酵素Q10を含有する油状物質9.62ｇ（補
酵素Q10として2.40ｇ）を得た。これを２等分し
てｎ−ヘキサンに溶解し、一方を前記と同様にし
て細孔容積0.85ml／ｇ（表面積450m2／ｇ）とな
るよう調製したシリカゲル（メルク社製）24ｇを
充填したカラムに負荷し、同じ溶媒で溶出して補
酵素Q10画分1.19ｇ（補酵素Q10として1.14ｇ）を
得た。純度95.8％、回収率95％であつた。残りの
半分を細孔容積0.75ml／ｇ（表面積500m2／ｇ）
となるよう調製したシリカゲル（メルク社製）24
ｇを充填したカラムに負荷し、同様の処理を施し
て補酵素Q10画分1.13ｇ（補酵素Q10として1.02
ｇ）を得た（回収率85.0％、純度90.2％）。これ
をアセトンにて三回晶析を行つたところ0.92ｇの
結晶が得られ純度は99.5％であつた。
細孔容積0.75ml／ｇ、0.85ml／ｇいづれの場合
も表１の結果とほゞ同じ純度および回収率を示し
た。細孔容積値を特定することによつて安定した
結果の得られることがわかる。
実施例 ２
補酵素Q9を生産するキヤンデイダ・トロピカ
リス（Candida tropicalis）IAM4924の培養液
100から菌体を分離し実施例１と同様に処理し
粗製補酵素Q9を27.28ｇ（CoQ9として6.08ｇ）得
た。これを四等分して、２％エーテル−ｎ−ヘキ
サンで洗浄溶出した点以外は実施例１と同様にク
ロマトグラフイー処理を行つた。その結果をまと
めると表２の通りである。[Table] Separately from the data in Table 1, Rhodotorula mucilaginosa AHU3946, which produces coenzyme Q 10 , was cultured for 20 days under the same conditions as in Table 1, and the same treatment was performed to produce an oil containing coenzyme Q 10 . 9.62 g of substance (2.40 g as coenzyme Q 10 ) was obtained. This was divided into two equal parts, dissolved in n-hexane, and one half was filled with 24 g of silica gel (manufactured by Merck & Co., Ltd.) prepared in the same manner as above to have a pore volume of 0.85 ml/g (surface area 450 m 2 /g). It was loaded onto a column and eluted with the same solvent to obtain 1.19 g of coenzyme Q 10 fraction (1.14 g as coenzyme Q 10 ). The purity was 95.8% and the recovery rate was 95%. The remaining half has a pore volume of 0.75ml/g (surface area of 500m 2 /g)
Silica gel (manufactured by Merck & Co., Ltd.) prepared so that 24
The coenzyme Q 10 fraction was 1.13 g (1.02 g as coenzyme Q 10 ) and subjected to the same treatment.
g) was obtained (recovery rate 85.0%, purity 90.2%). When this was crystallized three times with acetone, 0.92 g of crystals were obtained with a purity of 99.5%. Both the pore volumes of 0.75 ml/g and 0.85 ml/g showed approximately the same purity and recovery rate as the results in Table 1. It can be seen that stable results can be obtained by specifying the pore volume value. Example 2 Culture solution of Candida tropicalis IAM4924 producing coenzyme Q 9
Cells were isolated from 100 and treated in the same manner as in Example 1 to obtain 27.28 g of crude coenzyme Q 9 (6.08 g as CoQ 9 ). This was divided into four equal parts, and chromatography was performed in the same manner as in Example 1, except that the mixture was washed and eluted with 2% ether-n-hexane. The results are summarized in Table 2.
【表】
試料は、マススペクトル、逆相ペーパークロマ
トグラフイーから補酵素Q9であると同定された。
表２のデータとは別に、補酵素Q9を生産する
キヤンデイダ・トロピカリスIAM4924を同じ条
件で25培養し、同様の処理を行つて粗製補酵素
Q9を6.82ｇ（補酵素Q9として1.52ｇ）を得た。こ
れを細孔容積0.80ml／ｇ（表面積473m2／ｇ）に
調製したシリカゲル（メルク社製）24ｇを充填し
たカラムに負荷し表２の場合と同様に処理して補
酵素Q9画分1.47ｇ（補酵素Q9として1.37ｇ）を得
た（純度93.2％、回収率90.1％）。これをさらに
アセトンで３回晶析を行い純度99.0％の結晶1.23
ｇを得た。
細孔容積0.80ml／ｇの場合も、表２の結果と
ほゞ同じ純度および回収率を示し、本発明方法に
よつて優れた再現性、安定性の得られる事がわか
つた。
実施例 ３
乾燥酵母10Kgを実施例１と同様に処理し粗製補
酵素Q6を30.75ｇ（CoQ6として4.37ｇ）得た。こ
れを四等分して実施例１と同様にクロマトグラフ
イー処理を行つた。その結果をまとめると表３の
通りである。[Table] The sample was identified as coenzyme Q 9 by mass spectrometry and reversed phase paper chromatography. Separately from the data in Table 2, Candida tropicalis IAM4924, which produces coenzyme Q 9 , was cultured under the same conditions for 25 days and the same treatment was performed to produce crude coenzyme.
6.82 g of Q 9 (1.52 g as coenzyme Q 9 ) was obtained. This was loaded onto a column packed with 24 g of silica gel (manufactured by Merck & Co., Ltd.) prepared to have a pore volume of 0.80 ml/g (surface area 473 m 2 /g), and treated in the same manner as in Table 2 to obtain a fraction of coenzyme Q 9 of 1.47. (1.37 g as coenzyme Q 9 ) (purity 93.2%, recovery rate 90.1%). This was further crystallized three times with acetone, resulting in crystals of 1.23 with a purity of 99.0%.
I got g. Even when the pore volume was 0.80 ml/g, the purity and recovery rate were almost the same as the results in Table 2, indicating that the method of the present invention provides excellent reproducibility and stability. Example 3 10 kg of dry yeast was treated in the same manner as in Example 1 to obtain 30.75 g of crude coenzyme Q 6 (4.37 g as CoQ 6 ). This was divided into four equal parts and subjected to chromatography treatment in the same manner as in Example 1. The results are summarized in Table 3.
【表】
採取した補酵素Ｑはマススペクトル、逆相ペー
パークロマトグラフイーから補酵素Q6であるこ
とを確めた。[Table] The collected coenzyme Q was confirmed to be coenzyme Q 6 by mass spectrometry and reversed phase paper chromatography.
図１は表面積と細孔容積の関係を表わす図面で
ある。
FIG. 1 is a diagram showing the relationship between surface area and pore volume.
Claims (1)
クロマトグラフイーによつて精製する方法におい
て、異なる細孔容積値をもつシリカゲルを混合し
て当該粗補酵素Ｑ含有液の精製に最適の細孔容積
値をもつシリカゲルを調製し、これをクロマトグ
ラフイー用カラムに充填したカラムを使用してシ
リカゲル・カラム・クロマトグラフイーを行うこ
とを特徴とする補酵素Ｑの製造方法。 ２ 異なる細孔容積値をもつシリカゲルを混合し
て各種の細孔容積値をもつシリカゲルを調節し、
粗補酵素Ｑ含有液の補酵素Ｑ精製能を比較して最
適の細孔容積値を決める特許請求の範囲第１項記
載の製造方法。 ３ 最適値が0.80（ml／ｇ）〜0.90（ml／ｇ）の間
にある特許請求の範囲第１項もしくは第２項記載
の製造方法。 ４ 補酵素Ｑが補酵素Q6、Q7、Q8、Q9および
Q10よりなる群から選ばれたものである特許請求
の範囲第１項もしくは第２項記載の製造方法。 ５ シリカゲル・カラム・クロマトグラフイーの
展開溶媒が、炭素数５〜８の脂肪族炭化水素と炭
素を３〜６含むエステル、エーテルもしくはケン
トに該当する有機溶媒を組合せた混合溶媒である
特許請求の範囲第１項記載の製造方法。 ６ 異なる細孔容積値をもつシリカゲルを混合し
て粗補酵素Ｑ含有液の精製に最適の細孔容積値を
もつシリカゲルを調製し、当該シリカゲルをクロ
マトグラフイー用カラムに充填した補酵素Ｑ製造
用シリカゲルカラム。 ７ 粗補酵素Ｑ含有液の精製に最適の細孔容積値
が、0.80（ml／ｇ）〜0.90（ml／ｇ）の間にある特
許請求の範囲第６項記載のシリカゲルカラム。[Claims] 1. A crude coenzyme Q-containing solution is passed through a silica gel column.
In the method of purification by chromatography, silica gels with different pore volume values are mixed to prepare a silica gel with a pore volume value optimal for purifying the crude coenzyme Q-containing solution, and this is chromatographed. 1. A method for producing coenzyme Q, which comprises performing silica gel column chromatography using a column packed in a column for coenzyme Q. 2 Mix silica gels with different pore volume values to adjust silica gels with various pore volume values,
2. The production method according to claim 1, wherein the optimum pore volume value is determined by comparing the coenzyme Q purification abilities of the crude coenzyme Q-containing solutions. 3. The manufacturing method according to claim 1 or 2, wherein the optimum value is between 0.80 (ml/g) and 0.90 (ml/g). 4 Coenzyme Q is coenzyme Q 6 , Q 7 , Q 8 , Q 9 and
The manufacturing method according to claim 1 or 2, wherein the method is selected from the group consisting of Q10 . 5 The developing solvent for silica gel column chromatography is a mixed solvent consisting of a combination of an aliphatic hydrocarbon having 5 to 8 carbon atoms and an organic solvent corresponding to ester, ether or Kent containing 3 to 6 carbon atoms. The manufacturing method according to scope 1. 6 Coenzyme Q production by mixing silica gels with different pore volume values to prepare a silica gel with a pore volume value optimal for purifying a crude coenzyme Q-containing solution, and packing the silica gel into a chromatography column. silica gel column. 7. The silica gel column according to claim 6, wherein the pore volume value optimal for purifying the crude coenzyme Q-containing solution is between 0.80 (ml/g) and 0.90 (ml/g).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18950181A JPS5889193A (en) | 1981-11-25 | 1981-11-25 | Preparation of coenzyme q |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP18950181A JPS5889193A (en) | 1981-11-25 | 1981-11-25 | Preparation of coenzyme q |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5889193A JPS5889193A (en) | 1983-05-27 |
| JPS638759B2 true JPS638759B2 (en) | 1988-02-24 |
Family
ID=16242320
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP18950181A Granted JPS5889193A (en) | 1981-11-25 | 1981-11-25 | Preparation of coenzyme q |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5889193A (en) |
-
1981
- 1981-11-25 JP JP18950181A patent/JPS5889193A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5889193A (en) | 1983-05-27 |
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