JPH0113357B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for recovering useful substances by treating yeast cells that have accumulated useful substances.
More specifically, the present invention relates to a method for efficiently recovering useful substances from low-molecular-weight compounds excreted from the body through yeast cell treatment, which selectively excretes low-molecular-weight compounds present in the cytoplasm of the bacterial cells. Fermentation methods using yeast have long been widely known, and include, for example, amino acids such as glutamic acid and lysine, vitamins such as vitamin B ₂ and vitamin B ₁₂ , nucleosides, nucleotides, DNA, and RNA.
It is widely used as a method for producing nucleic acids such as and various enzymes. In the case of such methods, the target useful substance is generally accumulated within the bacterial body, so in order to recover the useful substance, it is necessary to crush the cells and separate the useful substance from the body. For this purpose, methods generally include mechanical grinding in a mortar, homogenizer, mixer, etc., lysis by autolysis, perchloric acid, sulfuric acid, formic acid, acetic acid, ethyl acetate,
Methods of treatment with chemicals such as acetone and toluene;
Methods such as treatment with cell wall lytic enzymes have been appropriately used. However, in these general separation methods, as the cell walls are destroyed during the treatment, not only the desired useful substances but also all the substances accumulated within the bacterial cells are excreted, and subsequent separation and purification operations are required. This method has the disadvantage that it is extremely complicated, and when using chemicals, it requires a neutralization step and causes decomposition of useful substances and toxicity. These problems can be solved at once if only the useful substances inside the microbial cells can be selectively separated from the body using safe substances.
Therefore, the present inventors conducted intensive studies to solve this problem, and found that when yeast cells are treated with an aqueous solution of divalent copper ions, only specific substances in the cytoplasm are selectively removed from the body. The present invention was completed based on the discovery that the substance is discharged. The purpose of the present invention is to efficiently extract target useful substances from low-molecular-weight compounds present in the cytoplasm by processing yeast cells that can separate intracellular components with safe and simple operations. We will provide a method for recovering it. Therefore, the method for treating yeast cells referred to in the purpose of the present invention involves contacting yeast cells that have accumulated useful substances within the cells with an aqueous solution of divalent copper ions, and expelling low-molecular compounds in the cytoplasmic fluid from the body. This is achieved by forcing people to do something. The yeast cells used in the present invention contain useful substances such as amino acids such as glutamic acid, lysine, aspartic acid, glutamine, homoserine, leucine, methionine, tryptophan, and threonine, vitamin B ₁ , vitamin B ₂ , vitamin B ₆ , vitamin _B12 , vitamins such as pantothenic acid, nucleic acids such as nucleosides, nucleotides, oligonucleotides, DNA, RNA, etc., S-adenosyl-L-methionine (hereinafter referred to as
SAM), physiologically active substances such as glutathione, and proteins such as protease and DNA polymerase, as appropriate. Such yeast cells are not particularly limited by the production method, and can be used appropriately as long as they are cultured according to conventional methods. Specific examples of the bacteria used include Satucharomyces, Pichia, Hansenula, Schizosatucharomyces, Satucharomycodes, Hanseniaspora, Torulopsis, Candeida, Rhodotorula, Cloveromyces, and the like. Among them, enzymes belonging to the genus Satucharomyces, Hansenula, Quyandida, Curveromyces, and Schizosatucharomyces, especially enzymes belonging to the genus Satucharomyces, are prized. However, even if the treatment of the present invention is applied to microorganisms other than yeast, such as Escherichia coli, the intended purpose cannot be achieved. In the present invention, the bacterial cells in the culture solution are collected by a conventional method, washed if necessary, and then treated with an aqueous divalent copper ion solution. Such treatment only requires contacting the bacterial cells with an aqueous copper ion solution;
This treatment reduces the number average molecular weight of amino acids, lower peptides, vitamins, nucleosides, nucleotides, etc. present in the cytoplasm to 1000.
In particular, low molecular weight compounds of 500 or less are quickly excreted from the body, while DNA, RNA, proteins,
Polymer compounds such as polysaccharides, SAM, arginine, and other substances present in the liquid bubbles remain within the bacterial cells. Such a phenomenon observed in copper ion treatment is very specific, and it is not possible to treat other divalent metal ions such as Mg ⁺⁺ , Ca ⁺⁺ , Sr ⁺⁺ , Ba ⁺⁺ , Mn ⁺⁺ , Fe ⁺⁺ ,
When Co ⁺⁺ , Ni ⁺⁺ , Zn ⁺⁺ , Sn ⁺⁺ , Pb ⁺⁺ , and Hg ⁺⁺ are used, the effects of the present invention cannot be achieved. The conditions for treatment with an aqueous copper ion solution may be selected as appropriate depending on the purpose, but the treatment is usually carried out by suspending the bacterial cells in water or a buffer solution, then adding the aqueous copper ion solution and leaving or stirring. be exposed. The pH at this time is usually 5 to 7.5, preferably 5.5 to 7, and the bacterial cell content in the suspension is usually 1 to 50% by weight, preferably 5 to 30% by weight, based on the weight of wet bacteria.
Contact temperature and contact time are usually 0 to 50℃ for 10 minutes to 3
The time is preferably 30 minutes to 2 hours at 20 to 40°C. The source of copper ions used may be any copper ion compound in an aqueous solution, and specific examples thereof include cupric chloride, cupric bromide, cupric sulfate, cupric acetate, etc. . The amount of copper ions added is usually determined by the concentration of copper ions in the suspension.
The amount is 5 μM or more, preferably 10 to 50 μM. Useful substances may be recovered from discharged low-molecular compounds by conventional methods, such as ion exchange chromatography, gel filtration, electrophoresis, solvent precipitation, etc., as appropriate. According to the present invention, it is possible to efficiently separate low-molecular-weight compounds in bacterial cells from other substances by a highly safe and simple operation of copper ion treatment at a low concentration, and It is extremely easy to recover useful substances found in .
Furthermore, even if the desired useful substance remains within the bacterial cells, separation and purification can be carried out extremely efficiently since low molecular weight compounds that serve as contaminants have been removed in advance. The present invention will be explained in more detail with reference to Examples below. Note that parts in Examples and Comparative Examples are based on weight. Example 1 One part of bacterial cells (wet bacterial cells) obtained by culturing Saccharomyces. Cerevisiae X2180-1A cultured in YEPD medium (1% yeast extract, 2% bactopeptone, 2% glucose) was added to 100 parts of a 100 μM cupric chloride aqueous solution and suspended at 30°C.
Stir gently. After a predetermined period of time, the concentrations of nucleotides, amino acids, and proteins present in the aqueous solution were measured, and the ratio per 1 g of dried bacterial cells was calculated based on the concentrations. The results are shown in Table 1. The quantification of nucleotides is performed using ultraviolet light (260n).
Amino acids were determined by fluorescence method using fluorescamine, and proteins were determined by the Lowry method based on absorbance and high performance liquid chromatography. From the results in Table 1, it can be seen that nucleotides and amino acids are rapidly extracted by contacting with an aqueous copper ion solution, but proteins are hardly extracted.

[Table] Example 2 A test was conducted in the same manner as in Example 1 except that the concentration of the aqueous copper ion solution or the type of copper compound was changed, and the amount of extracted nucleotides was measured.
The results are shown in Table 2.

[Table] Comparative Example 1 A test was conducted according to Example 1, except that another divalent metal compound was used in place of cupric chloride, and the extraction status of nucleotides was investigated. In addition, 260nm
The absorbance of indicates the presence of nucleotides. The results are shown in Table 3.

[Table] From the results, it can be seen that some nucleotides are extracted when mercury ions are used, but almost no nucleotides are extracted in other cases. In addition, when the test example using cupric chloride is expressed using the same evaluation method as in Table 3, the results are 0.108 (0 minutes) and 0.901 (30 minutes).
minute), 1.067 (60 minutes), which shows that the extraction efficiency is extremely superior even when compared to the case where mercury ions are used. Example 3 A test was conducted in the same manner as in Example 1, except that the bacterial cells shown in Table 4 were used in place of the bacterial cells used in Example 1, and the amounts of extracted nucleotides and amino acids were measured. , the ratio per 1 g of dry bacterial cells was calculated. The results are shown in Table 4.

[Table] Example 4 500 parts of the bacterial cells used in Example 1 were suspended in 10,000 parts of a 500 μM cupric chloride aqueous solution, stirred at 30° C. for 30 minutes, and then the bacterial cells were removed. Next, the amount of nicotinamide adenine dinucleotide (NAD) contained in the collected extract was determined by an enzymatic method using alcohol dehydrogenase, and was found to be 0.212 parts. This extract was passed through a formic acid type strongly acidic ion exchange resin (Dowex 1X-8, manufactured by Rohm and Haas) to adsorb NAD, and then the effluent was
The sample was washed with water until the absorbance at 260 nm became 0.1 or less, and then NAD was eluted with a mixed aqueous solution of 4M formic acid and 0.2M ammonium formate. The thus obtained NAD eluate was sequentially purified by sublimation and freeze-drying, and as a result, 0.170 parts of NAD was obtained. Example 5 After stirring for 30 minutes in the same manner as in Example 1, the amount of NAD in the extract was quantified, and the extracted amount was 3.2 μmol per gram of dry bacterial cells. Also OD ₂₆₀
The relative purity of NAD in the material was 15.4%. Comparative Example 2 One part of the bacterial cells used in Example 1 was added to 100% of 1.5N perchloric acid.
After shaking and extracting at room temperature for 1 hour, the mixture was neutralized with potassium hydrogen carbonate to remove the precipitate of potassium perchlorate formed. Then in the extract
When NAD was quantified, the amount was 1g of dry bacterial cells.
5.0 μmol per substance, and the relative purity in the OD ₂₆₀ substance is
It was 2.2%. Reference example 1 Schlenk.F. et al.'s medium [Journal of Biological Chemistry (J.
Biol. Chem.) Vol. 229, p. 1037 (1957)].
Cerevisiae) SAM obtained by culturing IFO2044
Tris (hydroxymethyl) containing 10 parts of bacterial cells (wet amount) was washed twice with distilled water and adjusted to pH 6.4.
It was suspended in 100 parts of aminoethane-2-[N-morpholino]ethanesulfonic acid buffer (buffer concentration 10 mmolar). Next, cupric chloride was added to the solution at a concentration of 100 μM, and the mixture was gently stirred at 30° C. for 1 hour to expel low-molecular impurities from the cells, and then the cells were collected and washed twice with distilled water. After that, it was suspended in 50 parts of distilled water, left to stand at -20℃ for 5 hours, and then frozen at 20℃.
The resulting suspension was centrifuged to remove insoluble substances, and the content and purity of SAM contained in the supernatant was measured.The SAM content was 1.70 parts. The relative purity was 90.9%. The purity of SAM is determined by taking a portion of the test solution and developing it with two-dimensional paper chromatography.
A spot of SAM was detected, the SAM concentration of the test solution was detected using an ultraviolet detector, and it was calculated from the measurement of OD ₂₆₀ of the test solution using the following formula. SAM purity=SAM OD ₂₆₀ /OD ₂₆₀ ×100 As can be seen from this reference example, the treatment method of the present invention is also useful as a method for extracting substances such as SAM that remain in the microbial cells.

Claims (1)

[Claims] 1. Treat yeast cells that have accumulated useful substances within the cells with an aqueous solution of divalent copper ions to expel low-molecular compounds in the cytoplasm from the body, and then recover the useful substances in the low-molecular compounds. A method for recovering useful substances from characteristic yeast cells.