KR100313666B1 - Mutant strain THERMOACTINOMYCESSP.M1 (KCTC8864P) and method for producing high yield of protease using same - Google Patents

Abstract

The present invention relates to a method for producing a high yield of a variant strain Thermoactinomyces sp. M1 (KCTC 8864P) and a proteolytic enzyme using the same, more specifically, a glucose transport system is deleted to inhibit catabolic metabolism. (Cabolite repression) is characterized in that the method of producing a strain M1 strain of thermoactinomyces released and a high yield proteinase using the same, when applied to the fermentation process as the inhibition of catabolic metabolism is released It is possible to produce enzyme in high yield without inhibiting enzyme production by glucose in the medium, and also to increase the enzyme production by 2.1 times when producing protease using enzyme production medium. Enhancing fermentation yield during enzyme production has the effect of reducing the enzyme production cost.

Description

Variation strain Thermoactinomyces genus M1 (VCTC 8864P) and high yield production method of protease using the same

The present invention relates to a method for producing a high yield of a variant strain Thermoactinomyces sp. M1 (KCTC 8864P) and a proteolytic enzyme using the same, more specifically, a variant strain thermoactino lacking a glucose transport system The present invention relates to a genus M1 and a method for producing protease using the same in high yield.

Catabolite repression observed in the use of microorganisms' energy sources is a phenomenon in which enzyme activity is inhibited by the presence of catabolic metabolites which can be quickly used in microbial growth media during fermentation using microorganisms. When cultivated in a medium containing other energy sources, many microorganisms inhibit the synthesis of glycolytic enzymes involved in the use of other energy sources other than glucose (Secretary, Microbial and Industrial , 21, 262-269 (1995)). ). Meanwhile, Saccharomyces cerevisiae , Aspergilli Gram-negative bacteria (eg , E. coli, Klesiella pneumoniae, Salmonella typhimurium ), and Gram-positive bacteria (eg , Bacillus subtilis, Streptomyces coelicolor ) As one can observe the phenomenon of glucose repression, the microorganisms are known to have different mechanisms for this.

In E. coli, which has been the most studied for inhibiting catabolic metabolism, catabolic inhibition is known to occur through the transcriptional regulation of catabolic enzymes by the cAMP-CAP complex.

In addition, in the case of Gram-positive bacteria including Bacillus, it is known to inhibit the synthesis of enzymes related to the use of various sugars by inducer exclusion mechanism, but it is still not known exactly about the inhibition of catabolic metabolism. On the other hand, in Bacillus and actinomycetes, the catabolic inhibition is known to regulate the transcription of enzymes involved in the use or degradation of various sugars, enzymes in the TCA cycle, and enzymes involved in the initiation of spore formation (Hueck, CJ & Wolfgang). , H.,Mol. Microbiol., 15, 395-401 (1995).

On the other hand, many Gram-positive bacteria including Bacillus are used to produce many industrial enzymes such as α-amylase, proteolytic enzyme, xylanase, cellulase and cellobiase as strains having many advantages for industrial applications. . The enzymes are mostly inducers derived in the presence of a substrate, the biosynthesis of which is regulated by biosynthetic metabolic regulators such as catabolic inhibition and feedback inhibition by end products. Therefore, in order to increase the productivity of industrial enzymes from the above industrial strains, a technique for releasing the biosynthetic control mechanisms of the enzymes by mutating and producing constitutive enzymes must be developed.

In addition, the mutant strains released from the control mechanism can be used without the preference for various sugars, which are carbon sources in the complex medium, thereby maximizing the use of sugars in the medium, thereby optimizing the production of enzymes using cheap substrates. This can be achieved, and in the end, enzyme production costs can be reduced, thereby increasing the productivity of industrial enzymes.

Examples of improving the yield of enzyme production by releasing the enzyme biosynthesis regulator are as follows: Production of alpha-amylase using Bacillus cereus (Yoshigi, N. et al., Agric. Biol. Chem. , 52, 2365-2366 (1988)); Production of beta-glucanase using Bacillus (Kruger, S. et al., J. Gen. Microbiol. , 139, 2047-2054 (1993)); Production of xylanase using Bacillus subtilis (Srivastava, R. et al., Biotechnol. Lett. , 15, 847-852 (1993)); Proteolytic enzyme production using Bacillus licheniformis (Bierbaum, G. et al., Appl. Microbiol. Biotechnol. , 40, 611-617 (1994)).

On the other hand, Thermoactinomyces sp. E79 strain (KCTC 1445 BP, Korean Patent Publication No. 95-4707) is a strain that produces useful industrial enzymes, such as heat-resistant, alkali-resistant proteinase (Lee , Jung-Kee et al., Biosci.Biotech.Biochem. , 60, 840-846 (1996)). However, when the protein hydrolase is produced using the strain, its production is inhibited by the inhibition of catabolic metabolites by glucose in the medium and there is a problem. Therefore, novel mutant strains and proteins using the same that constitutively produce enzymes without the presence or absence of glucose in the medium for effective enzyme production in Thermoactinomyces sp. There is a need for a method for producing a hydrolase.

The inventors of the present invention, as a result of diligent research efforts to solve the above problems, to prepare a mutant strain in which the inhibition of catabolic metabolism is released in Thermoactinomyces sp., The mutant strain is a high yield of proteinase The present invention was completed by confirming the synthesis of.

Accordingly, it is an object of the present invention to provide a thermoactinomyces rapid mutant strain in which catabolic metabolism inhibition is released.

In addition, another object of the present invention to provide a method for producing a high yield of proteinase using the strain.

1 is a graph showing the production pattern of proteolytic enzymes in the glucose-producing enzyme production medium,

Figure 2 is a graph showing the production pattern of proteolytic enzymes in the enzyme production medium not added glucose,

Figure 3 is a graph showing the inhibition of enzyme production and inhibition according to glucose concentration,

4 is a graph showing intracellular transport of isotopically labeled glucose,

5 is a graph showing intracellular transport of isotopically labeled glucose analog 2-deoxyglucose.

The present invention is characterized by a thermoactinomyces sp. Mutant strain in which the glucose transport system is deleted and catabolite repression is released.

In addition, the present invention is characterized by another method for producing a high yield proteinase using the mutant strain.

The present invention is described in more detail as follows:

Biosynthesis of heat-resistant, alkaline-resistant proteolytic enzymes of E79 (KCTC 1445 BP) genus Thermoactinomyces is inhibited by catabolic metabolites when glucose is added to the medium. Artificial mutagenesis are performed to prepare catabolic metabolism inhibitory resistant strains that exhibit degrading enzyme activity. There are various kinds of mutagens that artificially induce mutations, but in the present invention, it is possible to increase mutation efficiency with N-methyl-N-nitro-N-nitrosoguanidine, a chemical mutagenesis agent that directly acts on intracellular DNA. In order to induce mutations by using a combination of UV, another mutagenesis agent.

On the other hand, mutant strain selection is carried out using 2-deoxyglucose, a glucose analogue, in the selection medium. Unlike 2-glucose, 2-deoxyglucose is an analogue of glucose and transported into cells through the glucose transport system and phosphorylated. It is no longer metabolized and accumulates within the cell and is also toxic to the cell, resulting in cell death. Therefore, colonies that grow and appear in the selective medium added with 2-deoxyglucose are likely to be mutant strains that do not transport 2-deoxyglucose into cells due to mutations in the glucose transport system.

In addition, the selection medium includes skim milk. The reason for using the scheme milk is that strains producing a large amount of a protein hydrolase, which is the target strain of the present invention, can be selected by identifying a large transparent ring formed by decomposing the scheme milk in the medium.

Therefore, the medium for screening the mutant of the present invention is a medium double-checked by 2-deoxyglucose and skim milk, which is damaged only by the mutant metabolism inhibitory to the production of protease by impairing the glucose transport system. It can be said that the medium can be specifically selected.

As described above, the mutant strains from which the inhibition of catabolic metabolism was released from the mutated strains were selected, and the mutant strains were designated as Thermoactinomyces sp. M1, which is a Korean depositary institution and the international treaty of the Budapest Treaty. The deposit was made on September 9, 1998 at the Genetic Resources Center in the Institute of Biotechnology, Korea Advanced Institute of Science and Technology, and was given the deposit number KCTC 8864P.

On the other hand, as a result of examining the glucose transport of M1 in the mutant thermoactinomyces, it was found that the inhibition of catabolic metabolism in the mutant strain M1 was the result of the deletion of the glucose transport system. Accordingly, the present invention M1 genus Thermoactinomyces is characterized by a lack of glucose transport system.

In the present invention, the method for producing proteolytic enzymes is carried out using the above-mentioned variant strain Thermoactinomyces M1, wherein the medium used is preferably a complex medium or enzyme production medium containing various carbon sources. M1 strain of the present invention strain thermoactinomyces is inhibited catabolic metabolism inhibition by glucose, the method of the present invention using the same can improve the protease yield.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1 Preparation of Variant Thermoactinomyces Genus M1

Biosynthesis of heat-resistant, alkaline-resistant proteolytic enzymes of E79 (KCTC 1445 BP) in Thermoactinomyces is inhibited by catabolic metabolites when glucose is added to the medium, so even if high concentrations of glucose are present in the medium Mutant strains were prepared according to the following method to isolate catabolic metabolism inhibitory resistant strains that exhibited degrading enzyme activity: First, the genus E79 of Thermoactinomyces was enzymatically produced (1.0% soyton, 2.0% soluble starch, 0.3% K ₂ HPO ₄ , 0.05% MgSO ₄ , pH 7.2) until OD ₆₀₀ = 1, and then the mutagenesis agent N-methyl-N-nitro-N-nitrosoguanidine 250 ㎍ final concentration / Ml was added and incubated for 1 hour at 50 ℃. The mutated cells were then collected by centrifugation, washed with 15 ml of saline, and then suspended with 2 ml of saline. Then, the suspended cells were placed in a petri-dish and irradiated with UV light, which is a mutagenic agent, for 5 minutes at a distance of 30 to 40 cm, followed by addition of 2% skim milk and 30 mM 2-deoxyglucose. It was plated on enzyme-producing agar medium and incubated at 55 ° C. for 36 hours. Then, two strains, which degrade the skim milk of the selection medium to form a large transparent ring, are selected as a catabolic inhibitor-resistant strain, and the selected two strains contain up to 5% glucose and 2% skim milk. Incubation for 36 hours in the enzyme production medium was finally selected one strain with higher metabolism inhibitory resistance.

Accordingly, the finally selected mutant strain was named Thermoactinomyces sp. M1, and it was assigned to the Genetic Resource Center in the Biotechnology Research Institute of Korea Institute of Science and Technology, which is a depository institution of the Republic of Korea and an international depository institution under the Budapest Treaty, 1998. It was deposited on 9 January, and was assigned accession number KCTC 8864P.

Example 2 Protease Production of Mutant M1 in Glucose-added Enzyme Production Medium

In order to investigate the proteolytic enzyme production of the mutant thermoactinomyces M1 in the enzyme production medium of Example 1 with glucose added, and to compare the enzyme production capacity with wild strain E79, the final concentration of glucose was 1%. In the added 2 L enzyme production medium, two strains were respectively cultured in liquid at 50 ° C. using 5 L fermenter, and the change in absorbance at 600 nm was measured at intervals of 4 hours to confirm the growth of the strain. The activity of the degrading enzyme was measured. The activity of the protease was determined as follows: First, 0.5 ml of culture supernatant taken from the fermentor at intervals of 4 hours in a substrate solution (0.6% Hamarsten casein in 10 mM Gly-KCl-KOH buffer solution, After adding 3 ml of pH 10.0) and reacting at 55 ° C. for 15 minutes, 3 ml of TCA solution (0.11 M TCA, 0.22 M sodium acetate and 0.33 M acetic acid) was added to stop the reaction. Subsequently, undigested casein was allowed to settle for 10 minutes at room temperature and then absorbance was measured at 275 nm. On the other hand, the titer of the enzyme was determined by the amount of enzyme that increases the OD ₂₇₅ value by 0.01 to 1 unit.

As shown in Figure 1, both wild strain E79 and mutant strain M1 showed similar growth rate, but wild strain E79 showed very weak protease activity, whereas mutant strain M1 had a large amount of inhibitory inhibition of enzyme production. The enzyme was produced, and the enzyme titer was 1.2 times higher than that of the enzyme production medium without glucose.

On the other hand, when the amount of glucose remaining in the medium during fermentation was investigated, E79 in the wild strain Thermoactinomyces was very active at the beginning of fermentation, and after 16 hours of cultivation, the residual glucose content in the remaining fermentation broth was less than 0.2%. In contrast, M1 in the mutant thermoactinomyces showed 0.8% residual glucose. The consumption rate of sugar in wild wine was almost consumed after 24 hours, whereas in mutant wine, glucose decreased slowly, and about 0.2% of glucose remained even after 48 hours incubation. Therefore, the release of catabolic metabolism in the mutant strain M1 was found to be due to the deficiency of the glucose transport system.

Example 3 High Production of Proteolytic Enzyme Using Mutant M1 in Enzyme Production Medium Without Glucose

In the normal enzyme production medium of Example 1 in which glucose was not added, 4 hours of liquid culture under the same culture conditions as in Example 2 using 2 L enzyme production medium to determine the proteolytic enzyme productivity of the mutant strain M1. The cell concentration and protease activity of the culture supernatant were measured at intervals. Cell concentration and protease activity were measured in the same manner as in Example 2.

As shown in FIG. 2, the mutant strain Thermoactinomyces M1 had a maximum enzyme activity of 196 U / ml after 36 hours of incubation, and the maximal activity of protease was 2.1 times higher than that of wild strain E79. Thus, the mutant strain M1 was found to be a mutant strain that produces enzymes with higher yield than wild strains as well as the release of catabolic metabolism suppressed by glucose.

Experimental Example 1: Confirmation of release of enzyme production inhibition by glucose

Inoculate 2% of the spawn grown overnight in a liquid medium added with glucose to a final concentration of 0.25% to 20% in the enzyme production medium (200 ml) of Example 1, incubate at 50 ° C. for 20 hours, and then centrifuge. Drying amount was measured, and the culture supernatant was taken to measure proteolytic enzyme activity. The activity of the proteolytic enzyme was measured in the same manner as in Example 2.

As shown in FIG. 3, in the wild-type E79 strain of Thermoactinomyces, the inhibition of catabolic metabolism inhibiting the production of protease by glucose was observed, but in the mutant strain M1, it was confirmed that the phenomenon was released. In detail, E79 of the wild strain Thermoactinomyces genus significantly decreased protease activity with increasing glucose concentration in the medium, and almost lost protease activity in the presence of 0.25% glucose. On the other hand, in the case of mutant strain M1, the activity of protease was maintained unchanged even in the presence of 20% glucose.

Experimental Example 2 Intracellular Transport of Isotopically Labeled Glucose and Glucose Analogs

To investigate whether the release of catabolic metabolism found in the mutant strain M1 of Thermoactinomyces is due to the loss of glucose transport system, we performed the pulse-chase labeling experiment using isotopically labeled glucose as follows. First of all, wild strain E79 and mutant M1 each were treated with 40 ml of SKM (0.2% (NH ₄ ) ₂ SO ₄ , 1.4% K ₂ HPO ₄ , 0.6% KH ₂ PO ₄ , added with 1% maltose). 0.1% Na ₃ citrate, 0.02% MgSO ₄ 7H ₂ O, and 1.0% casamino acid, pH 7.5) were incubated in the medium of the logarithmic phase, and the cells were collected by centrifugation and washed twice with physiological saline. Subsequently, the washed cells were suspended in 20 ml of SKM medium from which the carbon source was removed, and then incubated at 50 rpm for 250 minutes at 50 ° C. Next, incubate for 10 minutes with the addition of 10 mM unlabeled glucose and add 5 mCi of [ ¹⁴ C] glucose or 2-deoxy-D [1 ^-3 H] glucose and 2 ml of sample at regular intervals. Was taken. Then, 10 ml of cooled 20% unlabeled glucose was added to the 2 ml sample, centrifuged, washed three times with 20% glucose, and the amount of isotope carried into the cell was measured by a liquid scintillation counter. .

As shown in FIG. 4, wild strain Thermoactinomyces E79 continued to increase in the amount of glucose carried over time, and after 60 minutes, carried glucose of 100 nmol / mg DCW (dry cell mass). Mutant strain M1 was found to carry little glucose.

In addition, even when the transport capacity of 2-deoxyglucose was examined using 2-deoxy-D [1- ³ H] glucose as a glucose analog, as shown in FIG. While the transport was continued, it was found that the mutant strain M1 had a sharp decrease in the carrying capacity compared with the wild strain.

From the above results, it was confirmed that the release of catabolic metabolism inhibitory to glucose observed in the mutant strain M1 was attributed to a decrease in the intracellular glucose concentration due to the deletion of the delivery system involved in glucose transport.

As described and demonstrated in detail above, the present invention provides a strain of Thermoactinomyces sp. M1 (KCTC 8864P) in which catabolic inhibition was released. In addition, the present invention provides a method for producing high yield of proteolytic enzyme using the mutant strain. When applying the actinomycetes M1 of the present invention to the fermentation process for the production of proteolytic enzymes, glucose in the medium as the inhibition of catabolic metabolism inhibited enzyme production by glucose in the complex medium is released It is possible to produce enzyme in high yield without inhibiting enzyme production and also to increase enzyme production by 2.1 times when producing protease using enzyme production medium. The fermentation yield is improved to reduce the cost of enzyme production.

Claims (5)

Thermoactinomyces sp. M1 (KCTC 8864P). The method of claim 1, wherein the Thermoactinomyces sp. Thermoactinomyces sp. Thermoactinomyces sp. M1 (KCTC 8864P), characterized in that the mutant lacking the glucose transport system. In the method for producing a protein hydrolase by culturing the microorganisms in the medium, the microorganism is Thermoactinomyces sp. Thermophilinomyces sp. M1 (KCTC 8864P) characterized in that the high Yield preparation method. 4. The method of claim 3, wherein the medium is a glucose-containing complex medium or an enzyme-producing complex medium. Thermoactinomyces sp. ( Thermoactinomyces sp.) Ml (KCTC 8864P) Proteolytic enzyme, characterized in that prepared by culturing.