KR960001816B1 - Method for producing streptokinase and streptodornase - Google Patents

Abstract

New Streptococcus sp. CSKM 67881 (KFCC 10681) (I) and CSKL 10101 (KFCC 10682) (II) are screened from the mutants treated with NTG to Streptococcus sp. ATCC 12449. (I) or (II) is simultaneously cultivated at pH 6.8-7.2, 35-38 deg. C, 50-500 rpm agitation and 0.1-1 vvm aeration in the medium containing casein or Serratio peptidase hydrolyzed casein 0.75%, glutamine 0.01%, cysteine 0.005% and yeast extract 1-3% as nitrogen sources, over 8500 unit per milliliter of streptokinase and over 2000 unit per milliliter of streptodornase.

Description

Simultaneous preparation of streptokinase and streptodornase by fermentation

The present invention relates to a method for producing streptokinase and streptodonase in high yield, and cultures microorganisms capable of producing streptokinase and streptodonase in a nutrient medium containing glucose and casein as main raw materials, and in the culture medium. It is the accumulation of straptokinase and streptodonase.

As a method for producing streptokinase and streptodonase by conventional fermentation, the cultured Streptococcus sp is cultured in a nutrient medium as in US Pat. However, this method has a disadvantage in that the production cost is low because the titer (1200-2000 units per ml) of streptokinase accumulated in the fermentation broth is low.

Therefore, the present invention relates to a method for producing two enzymes of high titer at the same time by improving the production method of streptokinase and streptodonase by the conventional fermentation.

In general, the yield of fermentation depends on the production capacity of microorganisms, the composition and culture conditions of fermentation broth, and especially the composition of fermentation broth must be appropriately combined with materials necessary for microbial growth, that is, various factors necessary for growth and production. Naturally, it is possible to obtain the maximum effect, and also in the fermentation of streptokinase and streptodonase, the composition of the medium greatly influences the growth of cells of carbon source, nitrogen source and trace factors and the production of both enzymes.

In view of these conditions, the present inventors studied the preparation method of streptokinase and streptodonase by microbial mutant strains. High yield of streptokinase and streptodonase by adding appropriate amounts of casein, glutamine, cysteine, and tuna extract decomposed by proteolytic enzymes in a medium consisting of carbon source, nitrogen source and other trace components at a speed of 50 to 50 rpm and aeration rate of 0.1 to 1 vmv The present invention was completed by discovering that it can be accumulated and produced. Microorganisms CSKM 67881, CSKL 10101 used in the present invention has been deposited with the Korean spawn association (other number KFCC10681, KFCC10682), ATCC 12449 can be obtained from ATCC. The present invention is described in more detail by the following examples.

Example 1

Strains used: Streptococcus mutant strain CSKL 10101 (KFCC 10682)

Pre-culture: Brain Heart Infusion (Difco Co.) 37g / ι, pH 7.0

Species medium: 3% glucose, 0.5% ceratioppeptidase-treated casein, 0.1% magnesium sulfate, 0.75% potassium phosphate, 0.2% potassium diphosphate, yeast extract 1%, 1% tuna extract, 0.01% glutamine, cysteine 0.005 %, pH7.0

Foot medium: Glucose 5%, Ceratiopeptiptase-treated casein 0.75%, Magnesium sulfate 0.1%, Yeast extract 1%, Tuna extract 1%, Potassium phosphate 0.75%, Dipotassium phosphate 0.2%, Glutamine 0.01%, Yeast extract 1%, tuna extract 1%, cysteine 0.005%, pH7.0

Cultivation method: 25 ml of the pre-culture medium of the above composition in a 250 ml Erlenmeyer flask and autoclaved at 121 ° C. for 15 minutes, followed by incubating CSKL 10101 with S. a. The seed medium 1ι was placed in a 2.5ι small fermenter and then autoclaved at 121 ° C. for 15 minutes, and then inoculated with 20 ml of the pre-culture solution of the Erlenmeyer flask. Conduct.

25 ml of fermented broth prepared by the addition of casein decomposed by Cerathio peptidase to the fermentation broth were added to a 5ι fermenter, and then autoclaved at 121 ° C. for 15 minutes, followed by inoculation of the seed culture solution with 4% volume, respectively. Under the conditions, incubation for 10 hours while aeration stirring under conditions of agitation speed 100rpm, aeration amount 0.6vvm. At this time, the cell concentration in the culture medium was measured for absorbance (OD) at 600 nm using a spectrophotometer, and the titers of streptokinase and streptodornase were determined by the C.R. 433 (1948)) and the Akira Takedo method (Akira Taketo and Yoriko Taket, Z. Naturforsch. 38.107-111 (1983)).

Table 1. Enzyme titers of ceratioppeptidase treated casein concentration]

Example 2

In Example 1, the results of enzyme titer after culturing according to concentration change, treatment time change, protease type, and the like using casein and casein digested by casein as a nitrogen source of the medium were shown in Tables 2, 3, Table 4 and so on.

[Table 2. Enzyme titers by casein concentration]

Table 3. Enzyme Titers According to Cerathiopeptidase Treatment Hours]

[Table 4. Enzyme Activity by Casein Enzyme Type]

Example 3

In Example 1, the enzyme titer after fermentation broth was fixed to 0.75% of enzyme degradation casein, and the enzyme titer after culturing according to the concentration change of glutamine and cysteine is shown in Table 5.

[Table 5. Glutamin and Cysteine Enzyme Activity by Concentration]

Example 4

In Example 1, the enzyme hydrolysis casein concentration was 0.75%, glutamine and cysteine concentrations were fixed at 0.01% and 0.005%, respectively, and the enzyme titers after culturing with varying tuna extract concentrations are shown in Table 6.

[Table 6. Enzyme Activity According to Tuna Extract Concentration]

Example 5

In Example 1, enzyme enzyme casein concentration 0.75%, glutamine and cysteine concentrations in fermentation broth were fixed to 0.01% and 0.005, respectively, and the enzyme titers after culturing by changing the concentration of yeast extract are shown in Table 7.

[Table 7. Enzyme titer according to yeast extract concentration]

Example 6

In Example 1, the enzyme titer after culturing with varying agitation speed and aeration amount is shown in Table 8.

[Table 8. Enzyme Titers According to Changes in Agitation Rate and Aeration Rate]

Claims (3)

Streptococcus strain KFCC 10681 or KFCC 10682 is cultured in a nutrient medium to produce high titers of streptokinase and streptodonase. The method according to claim 1, wherein the nutrient medium contains casein, glutamine, cysteine, tuna extract, and yeast extract digested by casein or protease as a nitrogen source. The method according to claim 1, wherein the culture method comprises culturing at a stirring speed of 50 to 500 rpm and an aeration rate of 0.1 to 1 vvm.