JPH0761259B2 - Extraction method of yeast intracellular substance - Google Patents
Extraction method of yeast intracellular substanceInfo
- Publication number
- JPH0761259B2 JPH0761259B2 JP2270032A JP27003290A JPH0761259B2 JP H0761259 B2 JPH0761259 B2 JP H0761259B2 JP 2270032 A JP2270032 A JP 2270032A JP 27003290 A JP27003290 A JP 27003290A JP H0761259 B2 JPH0761259 B2 JP H0761259B2
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- Prior art keywords
- cells
- yeast
- amount
- salt
- osmotic shock
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、酵母菌を機械的に破砕する事なく、細胞内に
含まれるタンパク質、紫外吸収物質などの細胞内物質の
抽出方法に関し、酵母菌が生産するタンパク質、核酸、
酵素などを分離、精製するための前処理法として有効な
方法である。また、遺伝子組み替え技術により酵母細胞
内に生産されたタンパク性物質の抽出法としても利用可
能である。TECHNICAL FIELD The present invention relates to a method for extracting intracellular substances such as proteins and ultraviolet absorbing substances contained in cells without mechanically crushing yeast. Proteins, nucleic acids produced by bacteria,
This is an effective pretreatment method for separating and purifying enzymes and the like. It can also be used as a method for extracting proteinaceous substances produced in yeast cells by gene recombination technology.
一般に、酵母菌は少数の酵素タンパクを除いて培地中に
タンパク質を分泌しないことが知られている。このた
め、酵母菌からタンパク質を得るためには、ガラスビー
ズと共に高速で振盪し、機械的に細胞を破砕した後、遠
心分離などにより菌体破砕物を除いた後、上清に含まれ
るタンパク質を分離、精製する方法が用いられている。
また、遺伝子組み替え技術により生産させたタンパク性
物質を、菌体外へ分泌させるためには、遺伝子組み替え
により分泌性ペプチドと目的とするペプチドを結合した
融合ペプチドを作らせるようなDNA断片を作製し、このD
NAを発現させることにより融合ペプチドを菌体外に分泌
させるなどの方法が試みられ、この方法によって、ある
特定のタンパク性物質については菌体外へ分泌させるこ
とが可能となっている。It is generally known that yeast do not secrete proteins into the medium except for a few enzyme proteins. Therefore, in order to obtain proteins from yeast, shake the cells at high speed with glass beads to mechanically disrupt the cells, remove the disrupted cells by centrifugation, etc., and then remove the proteins contained in the supernatant. A method of separating and purifying is used.
In addition, in order to secrete the proteinaceous substance produced by the gene recombination technology to the outside of the bacterial cell, a DNA fragment is prepared by which the secretory peptide is combined with the target peptide to form a fusion peptide. , This D
Attempts have been made to secrete the fusion peptide outside the cells by expressing NA, and this method makes it possible to secrete a specific proteinaceous substance outside the cells.
また、大腸菌を材料に、ヘッペル（Heppel）によって開
発された浸透圧ショック法により、ペリプラズム間隙に
局在する加水分解酵素などが抽出されることおよびここ
で抽出されるタンパク質量は細胞の全タンパク質の約４
％に相当することも報告されている（ジャーナル オブ
バイオロジカル ケミストリー誌（Journal of Biolo
gical Chemistry）第241巻3685頁（1965年）、同誌第24
1巻3055頁（1966年）、同誌第242巻2561頁（1967
年））。さらに、最近、遺伝子組み替え技術を用いて、
大腸菌の細胞内に生産されたヒトインターロイキン−１
βを、浸透圧ショック法により氷水中に漏出させる実験
（ジーン誌（Gene）第86巻,291頁（1990年））が報告さ
れている。In addition, hydrolyzing enzymes localized in the periplasmic space are extracted by the osmotic shock method developed by Heppel using E. coli as a material, and the amount of protein extracted here is the total protein of the cells. About 4
% Is also reported (Journal of Biological Chemistry).
gical Chemistry) Vol. 241, page 3685 (1965), Vol. 24.
Volume 1, page 3055 (1966), Vol. 242, page 2561, (1967)
Year)). Furthermore, recently, using genetic recombination technology,
Human interleukin-1 produced in E. coli cells
An experiment in which β is leaked into ice water by an osmotic shock method (Gene, Vol. 86, 291 (1990)) has been reported.
しかしながら、前記機械的に菌体を破砕する方法では、
機械的に破砕された細胞壁、細胞膜断片、細胞内顆粒が
タンパク質と混在しており、分離、精製を難かしくして
おり、また、融合ペプチドの菌体外に分泌させる方法に
おいても、菌体外には融合ペプチドとして分泌されるの
で、融合ペプチドから目的とするペプチドのみを取り出
すのに余分の手間を有し、簡便な方法とはいえないもの
であった。However, in the method of mechanically crushing bacterial cells,
Mechanically disrupted cell walls, cell membrane fragments, and intracellular granules coexist with proteins, making separation and purification difficult, and also in the method of secreting the fusion peptide extracellularly, Since it is secreted as a fusion peptide, it takes extra time to extract only the target peptide from the fusion peptide, and it cannot be said to be a simple method.
さらに、上記の浸透圧ショック法においては、大腸菌に
適用する場合の報告例は多々あるが、酵母菌に関する報
告は少なく、その上、報告されている方法（ヨーロピア
ン ジャーナル オブ バイオケミストリー（European
Journal of Biochemistry）第21巻137頁（1971年）
は、大腸菌で用いた方法に準じたものである。即ち、一
度培養した菌体を集菌洗浄後、高濃度の糖類などを含ん
だ培地で更にインキュベートした後、菌体を氷水また
は、氷冷した希簿マグネシウム溶液に、急激に再懸濁す
るというものである。しかし、大腸菌に比べて、酵母菌
は厚い細胞壁を有しているなど形態学的、分類学的に異
なっているため、この浸透圧ショック法は酵母菌に対し
て大腸菌などの効果を示さない。さらに、この方法は、
実験室レベルの少量の菌体を処理する方法としては扱い
易いが、実用的なレベルで大量の菌体を処理するには、
温度制御など操作の面から繁雑である。Furthermore, in the above-mentioned osmotic shock method, although there have been many reports of application to Escherichia coli, there are few reports on yeasts, and moreover, the method reported (European Journal of Biochemistry (European Journal)).
Journal of Biochemistry) Volume 21, Page 137 (1971)
Is based on the method used for E. coli. That is, after the cells that have been cultured once are collected and washed, they are further incubated in a medium containing a high concentration of saccharides, and then the cells are rapidly resuspended in ice water or an ice-cooled magnesium solution. It is a thing. However, the osmotic shock method does not show the effect of Escherichia coli and the like on yeasts because yeasts have morphologically and taxonomically different features such as having a thick cell wall as compared with Escherichia coli. Furthermore, this method
It is easy to handle as a method of treating a small amount of cells at the laboratory level, but to treat a large amount of cells at a practical level,
It is complicated in terms of operation such as temperature control.
そこで、本発明の課題は、酵母細胞から、安価で操作も
繁雑でない細胞内物質の抽出用を提供しようとする点に
ある。Therefore, an object of the present invention is to provide a method for extracting an intracellular substance that is inexpensive and is not complicated to operate from yeast cells.
味噌、醤油などの醸造に使われている耐塩性酵母は、約
18％の食塩存在下でも生育可能である（アドバンスズ
イン フード リサーチ）（Advances in Food Researc
h）第12巻53頁（1963年））。また、この酵母菌は、長
い間食品の生産菌として用いられてきたため、毒性物質
を生産することも無く、遺伝子組み替えの宿主として用
いても安全性の高い酵母菌である。そこで、この酵母菌
を供試菌株として、浸透圧ショックによる細胞内物質の
抽出に関して、pH条件、反応温度、薬剤添加、反応時
間、などを変えて浸透圧ショックをかけ、菌体内物質を
効率よく抽出させる方法を鋭意検討し、本発明を完成す
るに至ったものである。The salt-tolerant yeast used for brewing miso and soy sauce is about
It can grow even in the presence of 18% salt (Advance
In Food Research (Advances in Food Researc)
h) Volume 12, page 53 (1963)). Moreover, since this yeast has been used as a food-producing strain for a long time, it does not produce toxic substances and is a highly safe yeast even when used as a host for genetic recombination. Therefore, using this yeast strain as a test strain, when extracting intracellular substances by osmotic shock, osmotic shock is applied by changing pH conditions, reaction temperature, addition of drugs, reaction time, etc. The present invention has been completed by thoroughly studying the method of extraction.
すなわち、本発明は機械的に破砕する事なく、弱アルカ
リ性条件下で酵母菌に浸透圧ショックをかけることを特
徴とする細胞内物質を抽出する方法をその構成要件とす
るものである。That is, the present invention has as its constituent feature a method for extracting an intracellular substance characterized by subjecting a yeast to an osmotic shock under mildly alkaline conditions without mechanical crushing.
以下本発明を詳述する。The present invention will be described in detail below.
本発明の浸透圧ショック法を適用する酵母の種類は特に
限定されず、上記耐塩性酵母の他、耐塩性の低い酵母に
も適用できる。例えばアルコール発酵、パン製造などに
使用されるサッカロミセス セレビジェ（Saccharomyce
s cerevisiae）は、耐塩性酵母に比べて耐塩性が低く、
約８％の食塩存在下で生育が抑えられてしまう。しか
し、これら酵母についても本方法により、細胞内物質を
抽出することができる。The type of yeast to which the osmotic shock method of the present invention is applied is not particularly limited, and in addition to the salt-tolerant yeast described above, it can be applied to yeast having low salt tolerance. For example, Saccharomyce cerevisiae (Saccharomyce) used for alcoholic fermentation, bread production, etc.
s cerevisiae) has lower salt tolerance than salt tolerant yeast,
Growth is suppressed in the presence of about 8% salt. However, intracellular substances can also be extracted from these yeasts by this method.
本発明における浸透圧ショックは、例えばYPO培地等の
酵母用の培地に食塩を添加して酵母を培養し、これを集
菌した後食塩水で洗浄後、食塩を含まない弱アルカリ性
の緩衝液中に菌体を懸濁し、インキュベートすることに
より行う。The osmotic shock in the present invention is, for example, culturing yeast by adding salt to a medium for yeast such as YPO medium, collecting the bacteria and washing with salt solution, and then in a weak alkaline buffer solution containing no salt. It is carried out by suspending the cells in and incubating.
上記培地に添加する食塩の濃度は酵母の耐塩性いよって
異なり、その上限は酵母の生育を抑制しない範囲の濃度
にする必要があるが、例えばチゴサッカロミセス・ルキ
シー等の耐塩性酵母で使用する場合においては、培地中
の食塩濃度が約15〜16％程度が適当であり、また、サッ
カロミセスセレビジェ等の耐塩性のない酵母の場合は約
６〜７％程度が適当である。洗浄に使用する食塩水の濃
度は培地中の食塩濃度と同程度に調整する。また、緩衝
液のpHは、９〜10程度の弱アルカリ性が望ましく、緩衝
液の種類も特に限定されないが、例えば、トリス・塩酸
緩衝液、炭酸水素ナトリウム・炭酸ナトリウム緩衝液、
数％炭酸水素ナトリウム水溶液（重曹水）等が挙げられ
る。The concentration of the salt added to the medium depends on the salt tolerance of the yeast, the upper limit must be a concentration that does not inhibit the growth of yeast, for example when using salt-resistant yeast such as T. saccharomyces lucii In the above, the salt concentration in the medium is preferably about 15 to 16%, and about 6 to 7% in the case of yeast having no salt tolerance such as Saccharomyces cerevisiae. The concentration of the saline solution used for washing should be adjusted to the same level as the salt concentration in the medium. Further, the pH of the buffer solution is preferably weak alkaline of about 9 to 10, and the kind of the buffer solution is not particularly limited, but for example, Tris / hydrochloric acid buffer solution, sodium hydrogen carbonate / sodium carbonate buffer solution,
A few% sodium hydrogen carbonate aqueous solution (aqueous sodium hydrogen carbonate) and the like can be mentioned.
本発明の浸透圧ショックによる細胞内物質の抽出方法
は、機械的な菌体の破砕法等の従来の方法に比べて細胞
壁、細胞膜断片等の夾雑がなく、また簡便な操作で行う
ことができるほか、特殊な薬剤も使用しないものであっ
て、安価に大量の菌体を処理することができるととも
に、抽出される物質は食品衛生上等の安全性も高く、医
薬品工業あるいは食品工業における菌体処理手段として
極めて有用なものである。The method for extracting intracellular substances by osmotic shock of the present invention is free from contamination of cell walls, cell membrane fragments, etc., and can be carried out by a simple operation, as compared with conventional methods such as mechanical cell disruption. In addition, since it does not use any special chemicals, it can treat a large amount of cells inexpensively, and the extracted substance is highly safe in terms of food hygiene, etc. It is extremely useful as a processing means.
次に、実施例をあげながら、具体的にその方法について
説明する。Next, the method will be specifically described with reference to examples.
実施例１ 耐塩性酵母チゴサッカロミセス ルキシー（Zygosaccha
romyces rouxii）IFO1876株をYPD培地（酵母エキス１
％、ペプトン２％、グルコース２％）に15％食塩を添加
した培地（pH5）で培養した後、集菌し、15％食塩水で
洗浄後、pH4〜11の範囲の緩衝液に最終菌体濃度が５×1
07個/m1になるように懸濁する。30℃で１時間インキュ
ベートした後、遠心して菌体を除き、上清に抽出されて
くるタンパク質量、および、紫外吸収物質量を求めた。
タンパク質量は色素結合法（アナリティカル バイオケ
ミストリー（Analytical Biochemistry））第79巻544頁
（1977年））により、紫外吸収物質量は260nmの吸光度
より求めた。実験に用いた３種類の緩衝液は100mMクエ
ン酸・リン酸水素ニナトリウム緩衝液（pH4,5,6,7,
8）、100mMトリス・塩酸緩衝液（pH8,9）、100mM炭酸水
素ナトリウム・炭酸ナトリウム緩衝液（pH9,10,11）で
ある。Example 1 Salt-tolerant yeast Zygosaccha
romyces rouxii) IFO1876 strain in YPD medium (yeast extract 1
%, Peptone 2%, glucose 2%), cultured in a medium (pH 5) containing 15% sodium chloride, collected, washed with 15% saline, and then added with a buffer solution having a pH range of 4 to 11. Concentration is 5 × 1
Suspend at 0 7 cells / ml. After incubating at 30 ° C. for 1 hour, the cells were removed by centrifugation, and the amount of protein extracted into the supernatant and the amount of ultraviolet absorbing substance were determined.
The amount of protein was determined by the dye binding method (Analytical Biochemistry, Vol. 79, p. 544 (1977)), and the amount of ultraviolet absorbing substance was determined from the absorbance at 260 nm. The three types of buffer used in the experiment were 100 mM citric acid / disodium hydrogen phosphate buffer (pH 4,5,6,7,
8), 100 mM Tris / hydrochloric acid buffer (pH 8,9), 100 mM sodium hydrogen carbonate / sodium carbonate buffer (pH 9,10,11).
これらの結果、弱アルカリ性（pH9,10）の条件下で多量
のタンパク質が細胞内から抽出されてきた。トリス・塩
酸緩衝液（pH9）と炭酸水素ナトリウム・炭酸ナトリウ
ム緩衝液（pH9）の抽出効果を比べると、前者の方が少
し勝っていた。しかし、抽出物の食品などへの利用を考
えると、炭酸水素ナトリウム（重炭酸ナトリウム）と炭
酸ナトリウムは安全性が高く、安価であり、容易に入手
し易いことなどの理由から100mM炭酸水素ナトリウム・
炭酸ナトリウム緩衝液（pH9）を抽出溶液として、さら
に抽出条件を検討した。尚、ここでは、緩衝液を使用し
たが、数％の炭酸水素ナトリウム水溶液（重曹水、５％
水溶液でpH8付近）は弱アルカリ性であり、食品添加物
としても使用されており、その安全性は確認済みである
が、これを代用しても一向にさしつかえない。 As a result, a large amount of protein has been extracted from the cells under weakly alkaline conditions (pH 9, 10). Comparing the extraction effects of Tris / hydrochloric acid buffer (pH 9) and sodium hydrogen carbonate / sodium carbonate buffer (pH 9), the former was a little better. However, considering the use of the extract in foods, etc., sodium bicarbonate (sodium bicarbonate) and sodium carbonate have high safety, are inexpensive, and are easily available.
The extraction conditions were further examined using sodium carbonate buffer (pH 9) as the extraction solution. Although a buffer solution was used here, a few% sodium hydrogen carbonate aqueous solution (sodium bicarbonate solution, 5%
It is weakly alkaline (pH around 8 in aqueous solution) and is also used as a food additive, and its safety has been confirmed, but it can be used in any way even if it is substituted.
実施例２ 実施例１と同様な方法で培養、集菌、洗浄した菌体を最
終濃度５×107個/m1になるよう100mM炭酸水素ナトリウ
ム・炭酸ナトリウム緩衝液（pH9）に懸濁し、10,20,30,
40℃で１時間インキュベートし、その上清に抽出されて
くるタンパク質量と紫外吸収物質量を実施例１）と同様
な方法で求めた。Example 2 The cells cultured, collected and washed in the same manner as in Example 1 were suspended in 100 mM sodium hydrogen carbonate / sodium carbonate buffer (pH 9) so that the final concentration was 5 × 10 7 cells / m 1, and , 20,30,
After incubating at 40 ° C. for 1 hour, the amount of protein extracted into the supernatant and the amount of ultraviolet absorbing substance were determined by the same method as in Example 1).
タンパク質および紫外吸収物質は、共に、30℃における
抽出が良好であった。さらに30℃付近で抽出が良いこと
より、これは室温での抽出が可能となることを示してい
る。 Both protein and UV absorber were extracted well at 30 ° C. Furthermore, since the extraction is good at around 30 ° C, this indicates that the extraction can be performed at room temperature.
実施例３ 実施例１と同様な方法で培養、集菌、洗浄した菌体を最
終濃度が５×107個/m1になるよう100mM炭酸水素ナトリ
ウム・炭酸ナトリウム緩衝液（pH9）に懸濁し、その上
清に抽出されてくるタンパク質量と紫外吸収物質量を細
胞に含まれる全量と比較した。即ち、ガラスビーズ法に
よって機械的に破砕し、遠心分離により菌体破砕物を除
いた後、その上清に含まれるタンパク質量と紫外吸収物
質量を浸透圧ショックにより抽出される量と比較した。
尚、ガラスビーズ法により酵母細胞は100％近く破砕さ
れると考えられている。Example 3 In the same manner as in Example 1, the cells cultured, collected and washed were suspended in 100 mM sodium hydrogen carbonate / sodium carbonate buffer (pH 9) so that the final concentration was 5 × 10 7 cells / m1, The amount of protein extracted into the supernatant and the amount of ultraviolet absorbing substance were compared with the total amount contained in the cells. That is, the cells were mechanically disrupted by the glass beads method, the disrupted bacterial cells were removed by centrifugation, and the amount of protein and the amount of ultraviolet absorbent contained in the supernatant were compared with the amount extracted by osmotic shock.
It is believed that nearly 100% of yeast cells are crushed by the glass bead method.
浸透圧ショック法により抽出されてくるタンパク質量と
紫外吸収物質量は、機械的破砕法により得られる量の各
々、約34％および33％に達していた。これは、大腸菌に
おける浸透圧ショック法による全タンパク質の約４％に
比べて多量であり、菌体を破砕すること無く相当量のタ
ンパク質、紫外吸収物質を得ることが可能である。酵母
細胞内に生産されているタンパク質、核酸、酵素などを
抽出する方法として有効なものであると考えられる。 The amount of protein extracted by the osmotic shock method and the amount of UV absorbing substance reached about 34% and 33% of the amount obtained by the mechanical disruption method, respectively. This is a large amount compared to about 4% of the total protein in Escherichia coli by the osmotic shock method, and it is possible to obtain a considerable amount of protein and an ultraviolet absorbing substance without crushing the cells. It is considered to be effective as a method for extracting proteins, nucleic acids, enzymes, etc. produced in yeast cells.
参考例１ 実施例１と同様な方法で培養、集菌、洗浄した菌体を最
終濃度が５×107個/m1になるよう100mM炭酸水素ナトリ
ウム・炭酸ナトリウム緩衝液（pH9）に懸濁し、抽出溶
液に、大腸菌の場合に用いられているキレーターである
エチレンジアミン四酢酸二ナトリウム塩、金属イオンの
マグネシウムイオン、SH試薬である２−メルカプトエタ
ノールを加えて抽出を検討した。即ち、0.5mMエチレン
ジアミン四酢酸二ナトリウム二水塩、0.5mM塩化マグネ
シウム、あるいは1.0mM2−メルカプトエタノールを100m
M炭酸水素ナトリウム・炭酸ナトリウム緩衝液（pH9）に
添加し、その抽出に対する添加効果を検討した。Reference Example 1 In the same manner as in Example 1, the cells cultured, collected and washed were suspended in 100 mM sodium hydrogen carbonate / sodium carbonate buffer (pH 9) so that the final concentration was 5 × 10 7 cells / m1, Extraction was examined by adding ethylenediaminetetraacetic acid disodium salt, which is a chelator used in the case of Escherichia coli, magnesium ions of metal ions, and 2-mercaptoethanol, which is an SH reagent, to the extraction solution. That is, 0.5 mM ethylenediaminetetraacetic acid disodium dihydrate, 0.5 mM magnesium chloride, or 1.0 mM 2-mercaptoethanol was added to 100 m.
It was added to M sodium hydrogen carbonate / sodium carbonate buffer (pH 9), and the effect of the addition on the extraction was examined.
タンパク質の抽出に関しては、0.5mMエチレンジアミン
四酢酸二ナトリウム二水塩でわずかな効果が見られた
が、紫外吸収物質では効果が見られなかった。また、紫
外吸収物質のみに関しては、0.5mM塩化マグネシウムで
効果が見られたがタンパク質の抽出は無添加より低下し
ていた。これらの結果、特に、薬剤の添加は必要ないと
考えられた。 With respect to protein extraction, a slight effect was observed with 0.5 mM disodium ethylenediaminetetraacetic acid dihydrate, but not with the ultraviolet absorber. As for the ultraviolet absorber alone, 0.5 mM magnesium chloride was effective, but the protein extraction was lower than that without addition. From these results, it was considered that the addition of a drug was not particularly necessary.
実施例４ アルコール発酵、パン製造などに使用されるサッカロミ
セス セレビシェ（Saccharomyces cerevisiae）は、耐
塩性酵母に比べて耐塩性が低く、約８％の食塩存在下で
生育が抑えられる。この酵母をYPD培地（酵母エキス１
％、ペプトン２％、グルコース２％）に６％食塩を添加
した培地（pH5）で培養した後、集菌し、６％食塩水で
洗浄後、菌体を最終濃度が５×107個/m1になるよう100m
M炭酸水素ナトリウム・炭酸ナトリウム緩衝液（pH9）に
懸濁し、その上清に抽出されてくるタンパク質量と紫外
吸収物質量を細胞に含まれる全量と比較した。即ち、ガ
ラスビーズ法によって機械的に破砕し、遠心分離により
菌体破砕物を除いた後、その上清に含まれるタンパク質
量と紫外吸収物質量を浸透圧ショックにより抽出される
量と比較した。Example 4 Saccharomyces cerevisiae, which is used for alcoholic fermentation, bread production, etc., has lower salt tolerance than salt-tolerant yeast, and its growth is suppressed in the presence of about 8% salt. This yeast was mixed with YPD medium (yeast extract 1
%, Peptone 2%, glucose 2%), the cells were cultured in a medium (pH 5) containing 6% sodium chloride, and the cells were collected and washed with 6% saline, and the final concentration of the bacterial cells was 5 × 10 7 cells / 100m to be m1
The suspension was suspended in M sodium bicarbonate / sodium carbonate buffer (pH 9), and the amount of protein extracted into the supernatant and the amount of ultraviolet absorbing substance were compared with the total amount contained in the cells. That is, the cells were mechanically disrupted by the glass beads method, the disrupted bacterial cells were removed by centrifugation, and the amount of protein and the amount of ultraviolet absorbent contained in the supernatant were compared with the amount extracted by osmotic shock.
アルコール発酵、パン製造などに使用される酵母細胞か
らも、弱アルカリ性条件下の浸透圧ショックによりタン
パク質および紫外吸収物質が抽出された。しかし、耐塩
性酵母を用いた場合に比べて、浸透圧差が小さいこと、
細胞壁および細胞膜の構造に差異があるためか抽出量は
少なく、浸透圧ショック法により抽出されてくるタンパ
ク質量と紫外吸収物質量は、機械的破砕法により得られ
る量の各々、約１％および約３％であった。 Proteins and ultraviolet absorbing substances were also extracted from yeast cells used for alcoholic fermentation, bread making, etc. by osmotic shock under weak alkaline conditions. However, the difference in osmotic pressure is small compared to the case of using salt-tolerant yeast,
The amount of protein extracted and the amount of UV-absorbing substance extracted by the osmotic shock method are about 1% and about 1%, respectively, due to the difference in the structure of the cell wall and the cell membrane. It was 3%.
Claims (1)
件下で酵母菌に浸透圧ショックをかけることを特徴とす
る細胞内物質を抽出する方法。1. A method for extracting an intracellular substance, which comprises subjecting a yeast strain to osmotic shock under mildly alkaline conditions without mechanical disruption.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2270032A JPH0761259B2 (en) | 1990-10-08 | 1990-10-08 | Extraction method of yeast intracellular substance |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2270032A JPH0761259B2 (en) | 1990-10-08 | 1990-10-08 | Extraction method of yeast intracellular substance |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH04144677A JPH04144677A (en) | 1992-05-19 |
| JPH0761259B2 true JPH0761259B2 (en) | 1995-07-05 |
Family
ID=17480585
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2270032A Expired - Lifetime JPH0761259B2 (en) | 1990-10-08 | 1990-10-08 | Extraction method of yeast intracellular substance |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0761259B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6383810B2 (en) | 1997-02-14 | 2002-05-07 | Invitrogen Corporation | Dry powder cells and cell culture reagents and methods of production thereof |
| US8450099B2 (en) | 2005-09-16 | 2013-05-28 | Kyoto University | Process for producing thioredoxin by stressing yeast cells |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59109171A (en) * | 1982-12-14 | 1984-06-23 | Nippon Zeon Co Ltd | Treatment of yeast cell |
-
1990
- 1990-10-08 JP JP2270032A patent/JPH0761259B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH04144677A (en) | 1992-05-19 |
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