KR910007819B1 - Microorganism for producing streptokiuas & screptodprnase - Google Patents

Abstract

Streptokinase and streptodornase are produced by fermenting streptococcus sp. CSKM 67881 KFCC 10681 (I) or streptococcus sp. CSKL 10101 KFCC 10682 (II) in the medium contg. glucose, proteous casein, glutamine, cysteine, tuna ext., yeast ext., magnesium sulfate, etc.. The (I) and (II) are prepd. by treating the original strain streptococcus sp. lancefield's group C ATCC 12449 with NTG. The obtd. (I) or (II) produce streptokinase and streptodornase similtaneously.

Description

Microorganisms that produce streptokinase and streptodornase

The present invention relates to a microbial mutant strain that produces high titers of streptokinase and streptodonase, and is characterized in that cysteine availability is increased, and in skim milk, plasminogen-containing medium, Streptococcus mutant strains have the advantage of producing both high titers of streptokinase and streptodonase while forming a large clear zone.

Streptokinase activates plasminogen as plasmin to dissolve coagulated blood or protein factors, and streptokinase promotes depolymerization of nuclear proteins and nucleic acids. It is very important because it is used to relieve the symptoms of inflammation during surgery or infection.

Conventionally, as a preparation method of streptokinase and streptodonase, there is a method of separating and purifying the generated streptokinase after culturing microorganisms of Streptococcus in a nutrient medium as in US Pat. No. 2,701,227. There was a disadvantage in that the production cost was low due to the low titer of accumulated streptokinase (1200-2000 units per ml).

Therefore, the present invention is characterized by the separation of microbial mutant strains capable of simultaneously producing high titers of streptokinase and streptodonase.

The present invention is described in more detail as follows.

In other words, Streptococcus (Streptococcus sp. Lancefield's Group C ATCC 12449) strain obtained from the American Type Culture Collection (ATCC), an international microbial deposit institution, is based on the strain (NTG, N-metlyl-N'-). nitro-N-nitrosoguanidine) was treated with a mutagen to obtain mutant strains with significantly improved titers of streptokinase and streptodonase. Variation strain selection method is to treat the culture medium grown in the first medium to grow the bacteria in the second medium by treating the organism as an organic chemical mutant and colony (colon) appearing by smearing the milk on the third medium by digesting the skim milk (skim milk) Among the mutant strains with a large halo size, which is a clear zone, the mutant strains were separated according to colony size and colony morphological change, and streptokinase was compared to the double wild strain (ATCC 12449). And 3-4 fold higher streptodonase titers were named CSKM 67881 (KFCC 10681). This mutant has a titer of 4200-5400 units per ml of streptokinase and 1000-1200 units per ml of streptokinase in the fermentation broth. In addition, mutant strain CSKM 67881 was mutated according to the above-described method, and purely separated, and 7000-8500 units per ml of streptokinase in fermentation broth were 6-7 times higher in titer of streptokinase and streptodonase than wild strain (ATCC 12449). The mutant strains showing streptodonase titers of 1700-2000 units were selected and named CSKL 10101 (KFCC 10682).

Both microorganisms were deposited with the Korean spawn association and received accession numbers KFCC 10681 and KFCC 10682, respectively.

The culture characteristics of the mutants selected by the present inventors are milky color without producing a special pigment, glucose is used as the main carbon source, casein as the main nitrogen source, and the mutant is strepto when glutamine is added as a medium component. The kinase titer is increased and the streptodonase titer is increased when tuna extract is added. When cysteine is added to the media, the cell growth is greatly improved and the titers of streptokinase and streptodonase are increased. In addition, the use of casein digested with protease instead of casein usually significantly increases cell growth and increases streptokinase and streptodonase titers.

The strain characteristics, including the morphological features of the parent strain (ATCC 12449) and the variant strains of the present invention, are shown in Table 1.

TABLE 1

Physiological characteristics of the parent strain (ATCC 12449) and the mutants of the present invention are shown in Table 2.

TABLE 2

The enzyme titer was measured by the rover method (G.Rober and P. Kappus, Methods of Enzymatic Analysis Vol. 5, 433, 1984), and the titer of streptokinase was measured by the Akira Takedo method. Akira Taketo and Yoriko Z. Naturalforsch, 38 107-111 (1983)).

Note 1

Brain Heart Infusion (Difco co.) 37% pH 7.0

(Note 2) Enrichment Broth

Casein 1.0%, K ₂ HPO ₄ 1.0%, MgSO ₄ 7H ₂ O 0.1%, Yeast extract 0.1%, Juicy 0.1%, Tuna extract 0.1%, Glutamine 0.01%, pH 7.0

3) Mutant Screening Media

5.2% Brain Heart Infusion Agar, 1% skim milk, 0.001% plasminogen, pH 7.0

Note 4

Glucose 3%, Protease-treated casein 0.5%, Magnesium sulfate 0.1%, Potassium phosphate 0.75%, Dipotassium phosphate 0.2%, Yeast extract 1%, Tuna extract 1%, Glutamine 0.01%, Cysteine 0.005%, pH 7.0

(Note 5) fermentation medium

Glucose 5%, Proteolytic enzyme treated casein 0.75%, Magnesium sulfate 0.1%, Yeast extract 1%, Tuna extract 1%, Potassium phosphate 0.75%, Potassium phosphate 0.2%, Glutamine 0.01%, Cysteine 0.005%, pH 7.0

Example 1

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

All badges: 1 week

Species Badge: 4 Badges per Week

Fermentation medium: 5 week

Culture method:

25 ml of the above medium was dispensed into a 250 ml Erlenmeyer flask for shaking, autoclaved at 121 ° C. for 15 minutes, and then inoculated with the strain and incubated at 37 ° C. for 6 hours. 1L of seed medium was placed in a 2.5L small fermenter and autoclaved at 121 ° C. for 15 minutes, and then inoculated with 20 ml of the whole culture solution of the Erlenmeyer flask, and seeded at 37 ° C. for 6 hours under agitation speed of 100 rpm, air flow rate of 0.6vvm, and pH 7.0. do.

Add 2.5L of fermented broth prepared by adding enzyme-free casein and casein decomposed by Cerathio peptidase to each fermentation broth into a 5L fermentation tank and inoculated with 4% volume of seed culture solution after autoclaving at 121 ° C for 15 minutes. Incubate for 10 hours with aeration and agitation at conditions of stirring speed 100rpm, pH7.0, air flow rate 0.6vvm at 37 ℃. The cell concentration in the culture was measured for absorbance (O.D.) using a Spectrophotometer at 600nm. As a result of the culture, the use of enzyme-treated casein resulted in faster cell growth and higher titers of streptokinase and streptodonase.

TABLE 3

Example 2

As a result of changing the tuna extract concentration in Example 1, as the concentration of tuna extract increased, the streptokinase and streptodonase titer of the mutant strains improved.

TABLE 4

Example 3

As a result of investigating the effect of cysteine on ATCC 12449, mutant strains CSKM 67881 (KFCC 10681) and CSKL 10101 (KFCC 10682) as the strains used in Example 1, the mutant strains were faster than the parent strains and the titers of streptokinase and streptodonase were increased. The rise was high.

TABLE 5

Example 4

In the fermentation broth of Example 1, the titers of streptokinase and streptodonase of parent and mutant strains are shown in Table 6.

Claims (2)

Streptococcus strain CSKM 67881 (KFCC 10681), which is a strain of the genus Streptococcus characterized by producing streptokinase and streptodonase. Streptococcus strain CSKL 10101 (KFCC 10682), a strain of the genus Streptococcus characterized by producing streptokinase and streptodonase.