JPS5988087A - Production of cholesterol oxidase - Google Patents

Abstract

PURPOSE:To obtain cholesterol oxidase from the interior of a mold efficiently, by using an anionic surface active agent as a culture solution of a bacterium producing cholesterol oxidase. CONSTITUTION:An anionic surface active agent or it and bile acid or its salt are added to a culture solution obtained by cultivating a bacterium producing cholesterol oxidase (COD) in a growth medium or a solution suspending the mold collected from the culture solution, and cholesterol oxidase is extracted out of the mold. A bacterium producing and accumulating COD within the mold is used as the bacterium producing COD, for example, a bacterium belonging to the genus Streptomyces may be cited. COD extracted out of the mold is separated from the bacterium by centrifugation, or filtration, and obtained as a supernatant liquid or filtrate. COD is concentrated by ultrafiltration or salting out, so that the surface active agent used can be easily removed out of the system, and a product is prepared.

Description

Detailed Description of the Invention The present invention relates to a method for producing cholesterol oxidase (hereinafter abbreviated as 00D). 000D is a useful enzyme used for quantifying cholesterol in clinical tests. This method allows for colorimetric determination by directly injecting serum into a reagent to develop color, and has the advantages of being simple, sensitive, and highly accurate, and has seen remarkable popularity in recent years as an alternative to conventional chemical determination methods.

Hitherto, 00D has been known to originate from various microorganisms, and is broadly classified into those produced and accumulated outside the bacterial body and those produced and accumulated within the bacterial body. In the latter case, extraction operations such as grinding, ultrasonication, and bacteriolysis are required to extract 00D from the bacterial cells. The time and cost required for the operation are redundant compared to extracellular enzymes, which is one drawback.

In recent years, in order to overcome this drawback, a method for extracting COD from collected bacterial cells using a nonionic surfactant (Japanese Patent Application Laid-Open No. 49-1979) has been developed.
-47582 publication) or OOD by coexisting during culture.
Method for producing and accumulating extracellular bacteria (Japanese Patent Application Laid-Open No. 52-6128
8) has been developed. However, the surfactant is difficult to remove in a subsequent purification step and may interfere with the purification of 00D, so this method cannot be said to be sufficient.

Based on this background, the present inventors have diligently studied a simple and efficient extraction method with fewer engravings in the post-process, and have unexpectedly found that anionic surfactants have an essentially inhibiting effect on OOD. By using the agent at an appropriate concentration, A
The present inventors have discovered that OOD can be efficiently extracted from bacterial cells and that it is more effective to use bile acids or their salts in combination, and have completed the present invention. That is, the present invention provides an anionic surfactant alone or an anionic surfactant and a bile acid or its salt in a culture solution obtained by culturing OOD-producing microorganisms in a growth medium or in a suspension of bacterial cells collected from the culture solution. This is a method for producing 00'D, which is characterized by containing 00D and extracting 00D from the bacterial cells.

As the OOD-producing microorganism used in the present invention, one microorganism contains OOD.
There are microorganisms that produce and accumulate OD, such as those belonging to the genus Streptomyces.

Growth medium used in the present invention) The culture conditions are not particularly limited, and may follow conventional conditions.

In the present invention, the final concentration of anionic surfactant in the culture solution is preferably 0.02 to 0.2 as a final concentration% (vt/v).
% or in the presence of preferably 0.02 to 0.4% of bile acid or its salt and stirred at room temperature for about 30 minutes, OOD can be extracted out of the bacterial cells. In addition, when using collected bacterial cells, suspend the bacterial cells in an anionic surfactant or a solvent containing the surfactant and bile acid or its salt to the above concentration, and stir in the same manner to achieve the desired purpose. achieved. As anionic surfactants, sodium oleate 1 sodium dodecyl sulfate (hereinafter referred to as SD
S) and carboxylic acid salts such as sodium dodecylbenzenesulfonate, sulfuric acid ester salts, and sulfonic acid salts. Examples of bile acids or their salts include cholic acid, taurocholic acid, deoxycholic acid, glycocholic acid, and monoammonium salts of alkali metals such as sodium and potassium of these compounds. The OOD thus extracted outside the bacterial cells is sterilized by centrifugation or a transient method, and then obtained as a supernatant or p-liquid. If the OOD is concentrated by the ultra-transient method or the salting-out method, the used surfactant can be easily removed from the system and the product can be manufactured. In order to obtain a sample with even higher purity, it may be purified by chromatography using an ion exchanger or a molecular carrier. , it becomes possible to easily and efficiently extract OOD from bacterial cells without affecting subsequent processes.

Next, the method of the present invention will be specifically explained using an example using SDS as a typical example. A unit of 0 activity (TJ) is the enzyme titer that decomposes 1 micromole of cholesterol in 1 minute, which is measured by using 14-aminoantipyrine and 7 enol as a quinone imine dye in the presence of oxidase and measuring visible s (r+oonm). shall be.

Example 1 Soluble starch 1.5%, peptone 0.4%, yeast extract 0
．． 4%, meat extract 0.2%, calcium carbonate 0.2%,
Dipotassium phosphate 0.1% pMagnesium sulfate (7 hydrate) 0.05% m 11'eSO, (7 hydrate) 0.002
%, rice sugar white squeezed oil 1.0%, Adekanol LG-1260
, 2% (+) 87.2) containing 14 media.
Place the same composition medium (medium 50s
Streptomyces kenkabuensis (5tr
eptoqces capuensis) IPOl 3
024 (7) Inoculate the bacterial cells (14o-)L.

Aeration (1st/min) and stirring (AO) at 30°C for 30 hours.

r, p, m) were cultured. Using the thus obtained culture solution, an attempt was made to extract 00D inside the microbial cells using SDS alone or in combination with bile acid or its salt.

After addition of each surfactant (T)H'7.0), the mixture was stirred and centrifuged at room temperature for 30 minutes, and the OOD activity of the supernatant (extract) was measured. Table 1 shows the results. From Table 1, it is clear that when the culture medium contains SDS at an appropriate concentration, OOD is extracted efficiently, and when bile acid or its salt is coexisting, the extraction rate further increases and the extracted OOD is stabilized. It is permitted to do so.

Table 1 Example 2 Using the culture solution obtained in Example 1, the cells were collected by centrifugation and the same amount of 50mMK-! containing each surfactant was used. j
Resuspend the bacterial cells in acid buffer, I) H'7.0. Next, the mixture was stirred for 30 minutes at room temperature, centrifuged, and the COD activity of the supernatant (extract) was measured. The results are shown in Table 2.

Table 2 Patent Applicant: Toyobo Co., Ltd. Procedural Amendment 1 Indication of the Case 1982 Patent Application No. 197030 Title of the invention Process for producing cholesterol oxidase & Person making the amendment Relationship with the case Patent applicant Osaka City 2-2-8 Dojimahama, Kita-ku Table “Claims” and “Detailed Description of the Invention” columns of the specification to be amended (1) The scope of claims should be revised as shown in the attached sheet. Contents + 1 - (2) At the end of page 4 of the specification, the term "binary clause" is corrected to "molecular sieve."

(8) On page 5, line 2, "anionic surfactant" is corrected to "anionic surfactant."

(4) Page 5, line 13 Correct "in 1 minute" to "in 1 minute at 37°C".

(5) Page 6, line 1 Correct "rice bran white squeezed oil" to "rice bran white squeezed oil".

(6) Page 6, line 8 Correct "161/min" to "51/min".

(7) 2nd line from the bottom of page 7 Correct "contains the same amount" to "contains the same amount of culture solution."

2 - Attached Claim: ``A culture solution obtained by culturing cholesterol oxidase-producing microorganisms in a growth medium, or a suspension of bacterial cells collected from the culture solution, containing an ivy anion i surfactant or the surfactant and 3-480- A method for producing cholesterol oxidase, which comprises extracting cholesterol oxidase containing bile acid or a salt thereof out of bacterial cells." 3-480-

Claims (1)

[Claims] A culture solution obtained by culturing cholesterol oxidase-producing microorganisms in a growth medium or a suspension of bacterial cells collected from the culture solution is added with an anionic surfactant or the surfactant and bile acid or its salt to produce 1 cholesterol. 1. A method for producing cholesterol oxidase, which comprises extracting oxidase outside of bacterial cells.