JPH0759189B2 - Method for producing staphylokinase by Bacillus subtilis - Google Patents

Description

The present invention relates to a method for producing staphylokinase using a Bacillus subtilis host-vector system, and more specifically, to a Bacillus subtilis 168rec ^- mutation using a staphylokinase-producing gene as a host. A transformant obtained by transforming a Bacillus subtilis 168rec mutant strain with a recombinant plasmid incorporated in a plasmid vector of the strain is cultivated in an appropriate Bacillus subtilis medium to produce staphylokinase. Of staphylokinase by Bacillus subtilis characterized by adding at least one sugar selected from sugars consisting of sugar, glucose (fructose), fructose (fructose), sucrose (sucrose) and melibiose at a concentration of 0.25% or more. The present invention relates to a manufacturing method.

Staphylokinase referred to here (abbreviated as SAK below)
A, Staphylococcus aureus (Staphylococc
us aureus , hereinafter abbreviated as S. aureus), which is a plasminogen activator enzyme produced by the action of converting plasminogen to plasmin, and is thus known as anticoagulant or thrombosis agent. , Which can be used for medical purposes as a fibrous solvent.

Regarding the staphylokinase-producing gene (hereinafter referred to as sak gene) carrying the genetic information on the production of such SAK, this was extracted from the S. aureus lysogen phage Sφ-C and used as an E. coli plasmid vector. A system for efficiently producing SAK by cloning and expressing it in Escherichia coli as a host is disclosed in JP-A-58-6718.
No. 1 and JP-A-59-166085.

However, regarding Escherichia coli as a host, although its genetic and mycological properties are well known, when it is directed to the production of proteins such as SAK, the produced protein accumulates in the cells. Therefore, there is a problem in that the productivity is limited, and further, the protein is generally not easily purified, and there is a fear that a pyrogen is mixed.

Therefore, regarding the production of SAK, (1) Lysogen phage S
The φ-C-derived sak gene is realized by expressing it in the cells of S. aureus, which is a gram-positive bacterium, and (2) a gene that is generally expressed in a gram-positive bacterium is another gram-positive bacterium. Is likely to be expressed within
And (3) similar to Escherichia coli, in view of its capability of secretory production, its genetic and mycological properties are well known, but unlike E. coli, it is a Gram-positive bacterium. The host-vector system of Bacillus subtilis has been shown to be promising. Therefore, the present inventors have used the previously developed Bacillus subtilis host-vector system to incorporate it into one of the Bacillus subtilis plasmid vectors suitable for introducing the sak gene into Bacillus subtilis. When the transformant was introduced into Bacillus subtilis and the transformant was cultured, it was found that SAK was efficiently secreted and produced.

However, regarding Bacillus subtilis, the substance production performed by subjecting the strain to genetic manipulation has not yet reached the stage of practical application due to some problems. The problems are: (1) In most cases, it is difficult to stably retain the recombinant plasmid in the cells of Bacillus subtilis.

(2) It is difficult to artificially control the expression because analysis on the control of gene expression is insufficient.

(3) The substance produced is easily decomposed due to strong proteolytic enzyme (protease) activity.

And so on.

It was found that even in the SAK production system of the present inventors, a satisfactory production amount and stability cannot be obtained due to the reasons (2) and (3). The present inventors have result of extensive research for its improvement, Bacillus subtilis rec as Bacillus subtilis hosts ^-
By using a mutant strain and adding at least one sugar selected from glucose (glucose), fructose (fructose), sucrose (sucrose) and melibiose to the medium, no or little sugar is added. Compared to the case of using a medium supplemented with glucose (about 0.1%), not only the amount of SAK produced outside the cells is significantly increased, but also the degradation of the produced SAK is significantly suppressed. The present invention has been completed and the present invention has been completed.

That is, the object of the present invention is to have a sak gene,
In S. subtilis, a recombinant plasmid useful for producing SAK and Bacillus subtilis as a host (host-vector system of Bacillus subtilis) are used to efficiently produce SAK extracellularly. It is to provide a new manufacturing method of.

The configuration of the present invention in accordance with such an object is to produce a SAK by culturing a transformant obtained by transforming a Bacillus subtilis rec ^- mutant strain with a recombinant plasmid containing a sak gene in an appropriate culture medium. At that time, glucose (glucose) in the culture solution,
It is characterized in that at least one sugar selected from the sugars consisting of fructose (fructose), sucrose (sucrose) and melibiose is added. And regarding the Bacillus subtilis rec ^- mutant strain as a host which is one component of the present invention, preferably, Bacillus subtilis 168YIT-6007 (Bacillus subtil
is 168 YIT-6007), YIT-6008, and YIT-6009 are used (these Bacillus subtilis are each produced by Micro Inst.
No., 7464, and No. 7465 have been deposited with the Institute of Microbial Technology, Institute of Industrial Science and Technology).

Furthermore, pHSAK5, which is one of the preferred recombinant plasmids containing the sak gene, which is another component of the present invention.
65 is a composite plasmid pHY460 (Details of the composite plasmid are disclosed in JP-A-59-135891. E. coli (pHY460) as a transformant with the composite plasmid.
And Bacillus subtilis (pHY460) are respectively
No. 439 and Microtechnology Research Institute No. 439 have been deposited with the Institute of Microbial Technology, Institute of Industrial Science and Technology, and pSAK-HP2 (E. coli as a transformant with this plasmid (pSAK-HP2) is ATCC 3).
9179 (deposited with American Type Culture Collection as 9179) was enzymatically cleaved and ligated, and the sak gene derived from the S. aureus lysogen phage Sφ-C is contained in the gene (DNA). When the plasmid is introduced into Bacillus subtilis, it imparts SAK-producing ability and tetracycline resistance trait to the host bacterium, and provides an index (gene marker) for detecting and selecting transformants. . Then, in the production of a recombinant plasmid containing the sak gene, plasmid vectors that can be used include pUB110, pC194, and pHY60 in addition to the above-mentioned pHY460.
0 and derivatives thereof.

Furthermore, regarding the sugar which is the other one component of the present invention, the type is glucose (glucose), fructose (fructose), sucrose (sucrose), or melibiose, and the addition amount thereof is from 0.25% to A concentration range of 2.0% is preferred.

It was confirmed that the SAK-producing ability of Bacillus subtilis harboring the recombinant plasmid containing the sak gene was remarkably enhanced by adopting such a constitution.

That is, when culturing a transformant of Bacillus subtilis to produce SAK, when neither sugar is added to the culture solution or when a low concentration (0.1% or less) of sugar is added,
A considerable amount of SAK production was observed in the early stage of culture, but a sharp decline in SAK activity was confirmed thereafter. It is understood that this inactivation of SAK is caused by the degradation of SAK by the protease produced by Bacillus subtilis.

On the other hand, when any one of glucose (glucose), fructose (fructose), sucrose (sucrose) and melibiose was added to the culture medium, (1) SAK activity in the early stage of culture was not necessarily high. It was confirmed that the activity was rapidly increased in the mid-proliferation phase and that (2) it was difficult to be decomposed in the late proliferation phase.

The effects of sugar addition on SAK production will be described below by showing experimental examples.

<Experimental Example 1> Glucose (glucose) on SAK activity by Bacillus subtilis
The effect of added concentration was measured over time. That is, 50 ml of L-broth (Bacto-trypton) 1
%, Yeast extract 0.5%, NaCl 0.5%, pH 7.0) 0 to 2.0%
In a medium supplemented with glucose (glucose) at a concentration, transformants, Bacillus subtilis YIT-6007 (pHSAK56
5) was inoculated 1%, and cultured with shaking at 37 ° C., over time Kondo the SAK activity in the supernatant and Fujise method (Infect. 18. 26
6.1977). The results are as shown in Table 1.

Regarding the time-dependent changes in SAK activity, when glucose was added at a concentration of 0.5%, the activity was low in the early stage of culture, but showed a sharp increase in the middle growth phase, and reached a maximum value after about 16 to 19 hours. . Furthermore, when the protein in the culture solution collected over time was analyzed, the following items were confirmed.

(1) An increase in SAK protein is observed along with an increase in SAK production.

(2) Degradation is hardly observed until about 16 hours after the start of culture.

And from the measurement results shown in Table 1 and the above analysis results,
The amount of glucose added (concentration) is 0.25% to 2.0
%, Particularly preferably about 0.5% to 1.0%.

<Experimental Example 2> In this experimental example, the effect of adding various sugars on SAK activity was examined. The same method as in Experimental Example 1 was used except that the same basic medium as in Experimental Example 1 containing various sugars at a concentration of 0.5% was used. The results are shown in Table 2.

From these results, it was found that it was effective to add glucose (glucose), fructose (fructose), sucrose (sucrose) and melibiose as sugars.

Subsequently, reference examples and examples of the present invention will be described below.

Reference Example 1 Synthesis of recombinant plasmid pHSAK565 Composite plasmid pSAK-HP2 against composite plasmid pHY460
A DNA fragment containing the sak gene was cloned to synthesize a recombinant plasmid pHSAK565 (Tc ^r SAK ⁺ ).

That is, the composite plasmid p described in JP-A-59-135891.
Both DNAs of HY460 and the composite plasmid pSAK-HP2 incorporated into Escherichia coli deposited as ATCC 39179 were cleaved separately with restriction enzymes Bam HI and Pst I.
By mixing and ligating with T4 ligase,
A recombinant plasmid of 5.65 Md was synthesized (Fig. 1). This recombinant plasmid was named pHSAK565.

Reference Example 2 Transformation of Bacillus subtilis Bacillus subtilis (Bacillus subti) cultured overnight on an L-agar plate.
lis) 168HIT-6007 (Microtechnical Lab. No. 7326) was added to medium I (Spizizen minimal mediu)
m): K2HPO4 1.4%, KH2P04 0.6%, (NH4) 2 SO4 0.2
%, Sodium citrate 0.1%, MgSO4 / 7H2O 0.02%,
Casein hydrolyzate 0.02 for glucose 0.5%
%, Yeast extract 0.02%, and L-tryptophan 50 μg / ml) were inoculated at about 1 × 10 ⁸ / ml. This was shake-cultured at 37 ° C., and after about 4 hours, at the stage of entering the stationary phase, medium II (L-tryptophan in medium I
μg / ml, yeast extract 0.2%, 5 mM MgSO4 was further added, and diluted with 10 times in casein hydrolyzate) to continue the culture. After 90 minutes, the bacteria in the culture reached a competent state. The recombinant plasmid p synthesized in Reference Example 1 was added to 0.9 ml of this competent cell.
Transformation was performed while adding 0.1 ml of a DNA solution of HSAK565 and shaking culture at 37 ° C for 90 minutes. The transformant harboring the plasmid, Bacillus subtilis 168YIT-6007 (pHSAK565), has been deposited with the Institute of Microbial Science and Technology, National Institute of Advanced Industrial Science and Technology, as Microcosm Research Institute No. 7325.

〈Example〉 …… By Bacillus subtilis 168YIT-6007 (pHSAK565)
Production of SAK By the treatment described in Reference Example 2, transformant Bacillus subtilis 168YIT
6007 (pHSAK565) was obtained.

The SAK productivity of 50 transformant-resistant tetracycline strains was determined by the method of Kondo and Fujise (Infect.
mmun. 18. 266-272 (1977)), all strains were found to have SAK-producing ability. Furthermore, when the molecular weight of the plasmid carried by this transformant was measured,
Its molecular weight was about 5.65 Md.

Then, in a medium formed by adding 10 μg / ml of tetracycline to L-broth and 0.5% of glucose,
Bacillus subtilis 168YIT6007, which is the above transformant cultured overnight.
(PHSAK565) was inoculated to the above medium in an amount of 1/100, and the culture was continued at 37 ° C. with vigorous shaking.

After culturing for 17 hours, the SAK-producing ability in the culture supernatant obtained by removing the bacterial cells from the culture solution by centrifugation was measured by the fibrin plate method and found to be 50,000 units / m 2.
l SAK activity was confirmed.

This SAK activity was previously reported by Saito et al. (Journal of Bacteriology 38 (1) (1983)), plasmid pUBSF-.
It is about 5 times as large as that achieved by the method of introducing a recombinant plasmid in which the sak gene is incorporated into 13ΔH into Bacillus subtilis 168UOT-0734 (Microtechnology Research Institute No. 6872).

[Brief description of drawings]

Figure 1 shows the recombinant plasmid pH from pHY460 and pSAK-HP2.
FIG. 3 is a cleavage map showing a synthetic route to SAK565, and in the figure, sak
Represents the sak gene.

Claims (4)

[Claims] 1. A transformant obtained by transforming a Bacillus subtilis 168rec ^- mutant strain as a host with a recombinant plasmid comprising a staphylokinase-producing gene integrated into a plasmid vector of Bacillus subtilis 168 strain in a Bacillus subtilis medium. In the method for producing staphylokinase by Bacillus subtilis cultivated to produce staphylokinase, glucose (glucose), fructose (fructose), sucrose (sucrose) and melibiose are contained in the Bacillus subtilis medium. At least one sugar selected from sugars
A method for producing staphylokinase by Bacillus subtilis, which comprises adding at a concentration within the range of 25% to 2%. 2. A plasmid vector of Bacillus subtilis 168 strain is pUB
The method for producing staphylokinase by Bacillus subtilis according to claim 1, which is 110, pC194, pHY600, pHY460, or a derivative thereof. 3. The method for producing staphylokinase by Bacillus subtilis according to claim 1, wherein the recombinant plasmid is pHSAK565. 4. A Bacillus subtilis 168rec ^- mutant strain as a host is Bacillus subtilis 168YIT6007, Bacillus subtilis 168YIT6008, or Bacillus subtilis 16
The method for producing staphylokinase by Bacillus subtilis according to claim 1, which is 8YIT6009.