JPS61247380A - Production of enzyme - Google Patents

Abstract

PURPOSE:To efficiently separate and obtain the aimed enzyme, by reacting a protein modifying agent, etc., with a cell containing the aimed enzyme in the cytoplasm and a heterogenic enzyme in the cell membrane or cell wall, and selectively inactivating the heterogenic enzyme. CONSTITUTION:One or two or more selected from a protein modifying agent, acid, alkali, poassium chloride, organic solvent and surfactant are reacted with a cell containing the aimed enzyme in the cytoplasma and a heterogenic cell in the cell membrane or cell wall to inactivate selectively the heterogenic enzyme. The protein modifying agent, etc., are then removed to destroy the cell and the aimed enzyme is separated and obtained.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention efficiently separates and obtains a target enzyme from cells containing the target enzyme in the cytoplasm and a foreign enzyme in the cell membrane or cell wall. It is about the method.

[Conventional technology]

Conventionally, when obtaining the desired enzyme from such cells, the cells were first destroyed to obtain an enzyme extract, and then the foreign enzyme was extracted by gluchromatography.

The target enzyme is obtained by separating and removing it by appropriately combining known enzyme separation methods such as ion exchange chromatography, affinity chromatography, ammonium sulfate salting out, etc. [Problems to be solved by the invention] Separation of these is generally difficult, and as a result, these operating methods are combined in multiple stages, which not only increases the labor burden, but also results in low yields, which is a major problem.

[Means for solving problems]

The present invention solves these problems and provides a method for removing foreign enzymes contained in cell membranes or cell walls using simple means. That is, in the present invention, when separating and obtaining a desired enzyme from a cell whose cytoplasm contains the desired enzyme and whose cell membrane or cell wall contains a different enzyme, inactivating the foreign enzyme by acting with a protein denaturant, an acid, an alkali, potassium chloride, an organic solvent, or a surfactant;
The present invention relates to a method for producing an enzyme, which is characterized in that the target enzyme is separated and obtained by subsequently destroying the cells.

The target enzyme to be separated and obtained by the method of the present invention is an enzyme contained inside the cell, that is, within the cytoplasm. The type of such enzyme is not limited.

For example, aminoacylases such as N-benzoylglycine acylase, protocatechuic acid-3,4-dioxyke0
These include catalase, alcohol dehydrogenase, maleide dehydrogenase, cholesterol dehydrogenase, glycerol dehydrogenase, and glycerol kinase.

Aminoacylase is used, for example, in measuring the activity of carboxypeptidase A, but bacteria that produce this enzyme also produce peptidase. Contamination of aminoacylase with peptidase interferes with measurement of carboxypeptidase A activity. Although it is not easy to separate aminoacylase and peptidase using conventional separation and purification methods, peptidase can be easily removed using the method of the present invention that takes advantage of the difference in localized sites in cells.

The cell may be any cell that contains the desired enzyme in its cytoplasm, and includes various animal cells and plant cells in addition to microbial cells. An example of a cell is SchuP sonus putida flatus C684-2 (FERMP-
7716), protocatechuic acid-3,4- in the cytoplasm
Sheetmonas putida RB-4 (FEB) contains dioxygenase and peptidase in the cell membrane and cell wall.
M P-7369), Serratia marcescens IFO3054 containing catalase in the cytoplasm and gluconate dehydrogenase in the cell membrane, Pseudomonas aeruginosa IFO3445 and Hygluconopacter felicus I''
Examples include F012467.

When separating and obtaining the desired enzyme from such cells, the cells are first treated with a protein denaturant, an acid, an alkali, potassium chloride, an organic solvent, or a surfactant. These compounds deactivate foreign enzymes contained in the cell membrane or cell wall of cells containing the target enzyme in the cytoplasm, and are less likely to destroy cells.

Protein denaturants include, for example, urea and guanidine hydrochloride, and acids include sulfuric acid, hydrochloric acid, acetic acid, and pH 0.1 to 3 to 1.
Acidic buffers of about 0.0 IN and alkali include sodium hydroxide, potassium hydroxide, and alkaline buffers of about PHIO~12 and about t~0.0 IN. Potassium chloride is thought to function as a chaotropic effect or an electrostatic bond dissociator.

Organic solvents include ethanol, methanol, n-butanol,
Acetone etc. Surfactants are anionic (sodium dodecyl sulfate, etc.) and cationic (dodecyltrimethylammonium chloride, etc.).

There are nonionic (Triton X-100, etc.) and amphoteric (Amhitol, etc.) types, but any type can be used.

Those suitable for the purpose are selected depending on the cell, and it goes without saying that one should be selected that will not deactivate the target enzyme within the cell as much as possible. For example, urea is particularly suitable when obtaining aminoacylases.

It goes without saying that the above compounds are not limited to one type, but can be used in combination of two or more types as appropriate.

The cells are usually separated from the culture before being treated with the compound. Separation is generally performed by centrifugation, but other methods such as flocculation and sedimentation can also be used.

It is of course possible to allow the above-mentioned compounds to act without being separated from the culture.

In that case, the amount of compound used often increases.

After separation and before acting with the above-mentioned compound, if necessary, the cells are suspended and washed in water or a buffer solution.

The above compounds usually act on cells in a suspended state, but they can also act on cells in a slurry form.

The concentration of the compound and the action conditions such as temperature for 1 hour and pH are determined so as to deactivate the foreign enzyme in the cell membrane or cell wall as much as possible and not to deactivate the target enzyme.

This can be set by conducting a test for each cell in advance. For example, when treating Sheetmonas putida with urea, the appropriate concentration of urea is about O85 to IOM, preferably about 2 to 5M. Approximately 0.5 to 6 hours is appropriate.

After the above-mentioned compound has been allowed to act, it is usually removed. This removal is carried out, for example, by centrifuging to collect the cells and washing the cells if necessary. In some cases, such as organic solvents, it can be distilled off, and in other cases, it is only necessary to neutralize, such as acids and alkalis.

After removing the above compound, the cells are destroyed and the target enzyme is removed. Destruction of cells may be carried out by known general methods, such as Dyno Mill treatment and ultrasonic treatment.

Lysozyme treatment may be used.

The enzyme extracted from the cells is further purified, if necessary, by known enzyme purification methods such as r-chromatography, ion exchange chromatography, and ammonium sulfate salting out to obtain an enzyme preparation.

[Effect]

A protein denaturing agent for cells that contain the target enzyme in the cytoplasm and a foreign enzyme in the cell membrane or cell wall.

Foreign enzymes are selectively inactivated by the action of acids, alkalis, organic solvents, or surfactants.

〔Example〕

Example 1 Hippuric acid 1%, yeast extract 0.3%, sodium nitrate 0.
2%, dipotassium phosphate 0.1%, magnesium sulfate 7
Sheetmonas putida oboro 0684 was added to a pH 7.0 medium containing 0.05% aqueous salt and 0.05% sodium chloride.
-2 (FEBM P-7716) was inoculated and cultured with aeration for 12 to 17 hours while maintaining the temperature at 28 to 30°C. The culture solution was continuously centrifuged at 10.00 Orpm to collect the bacterial cells, and about 5ooII wet bacterial cells were obtained from the culture solution 601. The aminoacylase activity of this bacterial cell is 125.0OOU
The peptidase activity was 86.3U.

The wet bacterial cells were suspended in a 3M urea solution of 41 and stirred at room temperature for 1 hour. Next, the mixture was diluted with 8 to 10 times the volume of water, and the cells were collected by continuous centrifugation. The aminoacylase activity of this urea-treated bacterial cell was 125.0 OOU, and the peptidase activity was 11.3 U.

The above-mentioned urea-treated bacterial cells were suspended in buffer solution No. 31, and the cells were disrupted using a Dyno Mill and then centrifuged to remove cell debris. The resulting cell-free extract was subjected to DFAE-cellulose treatment, ammonium sulfate fractionation, and dialysis using conventional methods to obtain 260 Da of purified enzyme preparations with aminoarase activity of 76.0 OOU. The peptidase activity contained in this enzyme preparation was 0.5 U.

On the other hand, for comparison, Sheetmonas putida A 0684-2 (FERMIP
-7716), the cells were disrupted with pi guinomil and centrifuged to remove cell debris.

The aminoacylase activity of the obtained cell-free extract was 95.
00U, and the peptidase activity was 28.5U. This was subjected to DEAE-cellulose treatment and ammonium sulfate fractionation to obtain an enzyme preparation with amylase activity of 84,000 U and -e petiguse activity of 8.4 U.

Example 2 511 wet cells of Sheetmonas putida boiled 0684-2 (FERMP-7716) obtained by culturing in the same manner as in Example 1 were suspended in the following solution of 50- and stirred at room temperature for 1 hour. After that, the bacterial cells were collected in the same manner as in Example 1. The results of measuring the aminoacylase activity and peptidase activity of this bacterial cell are shown in the table below.

Aminoacylase Peptidase Activity Activity Untreated 643 1. I
X 1O-3n-butanol 273 0.
60X10-3 ethanol
203-IM KCl
466 0.33X10”-51M urea 5
68 0.30X10-30, OIM SPB
682 1. OX 10-' (pH 7,0) *1 Sodium phosphate buffer Example 3 Wet cells of Pseudomonas putida A C684-2 (FERN P-7716) obtained by culturing in the same manner as in Example 1 5 Ffr each , 50-, and after stirring at room temperature for 1 hour, the bacterial cells were collected in the same manner as in Example 1. The results of measuring the aminoacylase activity (white circles) and peptidase activity (black circles) of each bacterial cell are shown in FIG.

Example 4 p-hydroxybenzoic acid 1%, sodium nitrate 0.2%
, yeast extract 0.2%, dipotassium phosphate 0.1%
Pseudomonas putida RB-4 (FEBM P-7369) was inoculated into a pH 7.0 medium containing 0.05% sodium chloride and 0.05% magnesium sulfate heptahydrate, and cultured with aeration at 28°C for 18 hours. . The culture solution was continuously centrifuged at 10.00 Orpm to obtain wet bacterial cells.

Protocatechuic acid-3, which is an enzyme in the cytoplasm of this bacterial cell,
4-Dioxyrnase activity is 370 U per 11 wet bacterial cells.
The peptidase activity, which is a cell membrane and cell wall enzyme, was 3U.

This wet bacterial body 5 Ft-507! When suspended in a 3M urea solution and treated at room temperature for 1 hour, protocatechinic acid-
Peptidase activity could be reduced to 45% before treatment without inactivating 3,4-nooxidanase at all.

Example 5 Glucose 0.5%, peptone 0.5%, meat extract 0.
25%, sodium chloride 0.3%, magnesium sulfate 7
Serratia marcescens isolated from soil was inoculated into a pH 7.0 medium containing 0.05% aqueous salt and 0.05% dipotassium phosphate, cultured with aeration at 28°C for 18 hours, and moist bacterial cells were collected from the culture solution. was separated.

Catalase activity, which is an enzyme in the cytoplasm of this bacterial cell, is 230
U/I! It is a wet bacterial cell, and the activity of gluconate dehydrogenase (E, C, 1, 1, 99, 3), which is a cell membrane enzyme, is 47
ψIt was a wet bacterial body.

By treating 5.91 of these wet bacterial cells with 1.5M urea solution for 1 hour, we were able to reduce gluconate dehydrogenase to 20% of the pre-treatment level without inactivating catalase at all. .

〔Effect of the invention〕

According to the method of the present invention, a foreign enzyme that is difficult to separate from the target enzyme can be efficiently removed, the purification process can be greatly simplified, and the yield can also be improved.

[Brief explanation of the drawing]

FIG. 1 shows the relationship between urea concentration and enzyme activity after urea treatment. Patent applicant Fujirebio Co., Ltd. Agent Patent attorney 1) Masahiro Naka Figure 1 Kamenotomo (M)

Claims (1)

[Claims] When separating and obtaining the desired enzyme from cells that contain the desired enzyme in the cytoplasm and a different type of enzyme in the cell membrane or cell wall, the cell is first treated with a protein denaturing agent, acid, alkali, etc. A method for producing an enzyme, which comprises deactivating the foreign enzyme by applying potassium chloride, an organic solvent, or a surfactant, and then destroying the cells to separate and obtain the desired enzyme.