KR100776813B1 - A noble halophilic strain bacillus seohaeanensis bh724t which produces alkali-halophilic protease - Google Patents

Abstract

A novel strain Bacillus seohaeanensis BH724T is provided to be variously applied to an enzymatic industry for detergent and an enzymatic industry for food by showing significantly excellent productivity of a protease compared to a conventional bacillus strain and having both halophilic characteristics and alkalinity. A halophilic Bacillus seohaeanensis BH724T producing an alkali-halophilic protease is deposited as a deposition no. KCTC 11021BP and is obtained by shaking a sample taken from a saltern area in saline solution, painting a diluted solution over 10% agar medium, 7% sodium chloride and 1.5% agar and then culturing it at a temperature of 30 deg.C for 24-48 hours.

Description

A noble halophilic strain Bacillus seohaeanensis BH724T which produces alkali-halophilic protease

Figure 1 shows a plate photograph of Bacillus West Haenancesis BH724T.

Figure 2 shows a transmission electron microscope (TEM) of the Bacillus West Sea Annesis BH724T.

Figure 3 shows the phylogenetic tree of the strain of the present invention.

Figure 4 is added to 7% (w / v) NaCl medium (Marine agar, 3% NaCl) medium containing 10% skim milk to a final NaCl concentration of 10% (w / v), Bacillus West Haenensis Protease activity and growth status of BH724T (A-B-BH724T strains) strains of Bacillus subtilis KCTC 1028 strain (B-left of right and left plates) It is shown in comparison with Niformis KCTC 3049 ( Bacillus licheniformis KCTC 3049) strain (No. C on the left and right plates).

Figure 5 is a graph comparing the protease activity according to NaCl concentration of Bacillus West Haenensis BH724T with the patented Bacillus strain.

Figure 6 is a graph showing the change in protease activity according to the pH change of Bacillus West Haenancesis BH724T.

Figure 7 is a graph showing the change in protease activity according to the temperature change of Bacillus West Haenancesis BH724T.

The present invention relates to a basophilic Bacillus West Haenensis BH724T for producing a basophilic alkaline protease, and more specifically, the productivity of the protease is significantly superior to the existing Bacillus bacteria and has both a basophilic and alkaline properties at the same time The present invention relates to Bacillus seohaeanensis BH724T (KCTC 11021BP), which can be used in a variety of enzyme and food enzyme industries.

Microorganisms not only account for 60% of the world's biomass, but also account for more than half of the oxygen production, and they are at the core of the earth's ecology. It provides tremendous economic value that is indispensable. With the signing of the "Biodiversity Convention" at the United Nations Conference on Environment and Development in Rio de Janeiro in 1992, the growing awareness of the importance of biological resources around the world has led to the search for new microbes to secure diverse biological resources and develop high value added biological resources. Efforts are intensifying [Omura, The search for bioactive compounds from microorganism, Springer-Verlag, Tokyo, 1992]. The industrial enzyme market is more than $ 1 billion, of which the largest protease is more than 60%. Protease is a representative bioactive substance that manages various physiological functions. It is used for food, detergents, textiles, pulp and leather industry, and pharmaceuticals. In the past, proteases obtained from animals or plants were mainly used, but in recent years, they are produced in large quantities from microorganisms. Proteases are classified into alkaline, neutral and acidic proteases according to the optimum pH of enzyme activity.Alkaline proteases are mainly used in the detergent industry, including Bacillus licheniformis , Bacillus bacteria, Streptomyces bacteria, or Aspergillus. It is produced by molds such as Aspergillus oryzae . So far, the development has been mainly alkaline or high temperature strains, basophilic protease has not been developed yet. Recently released Bacillus amyloliquefaciens CMB01 strain, an alkaline protease producing strain [OH. BAN, SS. HAN, YN LEE, Annals of Microbiology, 53, 95-103 (2003)], a strain isolated from decayed rice porridge, is highly available as an enzyme-producing strain for detergents with amylase and lipase activity, but has little effect on salts. Therefore, the scope of application is limited. Therefore, the basophilic alkaline protease possessed by a strain isolated in an environment such as a salt farm containing a high concentration of salt can be used in a wide range of applications not only for detergent but also for food.

In view of the above, the present inventors have isolated the bacteriostatic bacillus Bacillus seohaenensis BH724T, which has an excellent alkaline protease activity compared to the existing Bacillus bacteria, to grow well at high salt concentrations using the same, and to produce protease. The present invention has been completed by succeeding in identifying these excellent characteristics.

Accordingly, it is an object of the present invention to provide a basophilic bacterium Bacillus westhaensis BH724T [KCTC 11021BP], which produces basophilic alkaline protease.

The present invention is characterized by a basophilic bacterium Bacillus westhaenensis BH724T [KCTC 11021BP], which produces basophilic alkaline protease.

Hereinafter, the present invention will be described in more detail.

The present invention is significantly superior in productivity of the protease compared to the existing Bacillus bacteria and has the characteristics of basophilic and alkaline properties at the same time the new strain Bacillus West Haenancesis BH724T that can be used in a variety of enzyme industry for food and enzyme industry for food ( Bacillus seohaeanensis BH724T) [KCTC 11021BP].

In the present invention, a sample was taken from the salted area to separate basophilic microorganisms, and 1 g of the sample was added to 9 ml of physiological saline (0.85% NaCl) and shaken for 30 minutes. Dilute the serial dilutions of this solution in 10 multiples in 10% opaque casein agar medium (skim milk, skim milk), 7% sodium chloride (NaCl) and agar (agar, 1.5%) and incubate at 30 ° C for 24 to 48 hours. The morphological, physiological and biochemistry of the strains of the present invention were selected according to partial sequencing of the strains of Casein's Manual of Determinative Bacteriology and 16s rRNA by screening strains of casein around the colonies. Strain identification was performed by examining the red and genetic characteristics.

As a result, it was classified as a new strain belonging to the genus Bacillus, and the strain of the present invention was named Bacillus seohaeanensis BH724T (International Journal of Systematic and Evolutionary Microbiology, 56 (8): p1893-1898, 2006), It was deposited with the Korea Biotechnology Research Institute Gene Bank, an international microorganism depositing institution, on November 7, 2006 and received accession number KCTC 11021BP.

In addition, a large amount of alkaline protease pMSL1 was obtained from basophils, and such activity is 2.6 to 12.4 times better than that of Bacillus.

Therefore, the new strain of the present invention is expected to be usefully used in the enzyme industry for detergents and the enzyme industry for food.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1 Isolation and Identification of Protease-producing Strains

Samples were taken from the salt farm area of the west coast of Taean-gun, Chungnam, and 1 g of the sample was added to 9 ml of physiological saline (0.85% NaCl) and shaken for 30 minutes. Dilute the serial dilutions of this solution in 10-fold multiples in opaque casein agar medium (skim milk), 10% and sodium chloride (NaCl) 7%, agar (agar, 1.5%) and incubate at 30 ° C for 24 to 48 hours. Strains that decompose casein around the colonies to form a clear ring were selected. As a control, Bacillus subtilis KCTC 1028 strain, which is known to have high productivity of protease, was used, and strains having better casein degradability than the control group were finally selected.

Highly casein-degrading strains were selected and re-incubated in marine agar medium (Marine agar), followed by secondary selection of high protease productivity using casein as a substrate.

L. B. Colonies grown on solid medium such as nutrient agar medium, marine medium, casein agar medium, and trypticase soy medium were observed.

The microbial growth curve, enzyme production curve, oxygen dependence, substrate consumption, catalase test, optimal growth temperature, optimal hydrogen ion concentration, and osmotic durability were examined to determine the physiological characteristics of the bacteria.

Biochemical tests included sugar degradation tests, methyl red and Vogs-Proskauer tests, citric acid utilization tests, urea degradation, indole and hydrogen sulfide production.

Isolated strains grow well in various Luria-Bertiani medium, nutrient agar, casein agar, trypticase soy, and Benett's agar. It became. The isolated strain was an aerobic bacterium, and growth was observed in the range of 15 to 50 ℃ and the optimum growth temperature was 35 to 40 ℃. On the other hand, growth was observed in the range of pH 5-9 and the optimum growth pH was around 8.0. The isolate was grown at 0-12% (w / v) salt (NaCl) concentration and the optimal salt concentration was 3-5%. In particular, active growth was observed in the range of pH 7.0-9.0, it is thought that the isolated strain prefers a neutral-alkaline environment. The isolate was Gram-positive bacillus, 0.5-0.6 × 1.3-1.8 μm in size, non-motility without flagella, spherical endospores, and spores in the center.

Physiological and biochemical properties of the BH724T strain are as follows.

Available Substrates: Glucose, Maltose, Trehalose, Xylose, Ribose, Glycerol, Mannitol, Fructose, Arbutin, Mannose, Casein, Asculin, Urea

○ Substrates not available: arabinose, rhamnose, lactose, adonitol, raffinose, salicylic acid, melibis, gelatin, starch, tyrosine, twin 80, xanthine, hypoxanthine

Osmotic durability: Grows at 20% sucrose and 12% NaCl.

○ biochemical properties

   Positive reactions: catarases, oxidases

Negative reaction: Nitrate reduction (NO ₃ → NO ₂ )

Morphological features were observed with a transmission electron microscope (TEM), as shown in FIG. 2.

Coenzyme Q was extracted and analyzed by high performance liquid chromatography (HPLC) using acetone-acetonitrile (80:20) as a developing solvent. Coenzyme Q of BH724 strain Was of type MK-7.

Cell fatty acids were extracted by saponication-methylation and analyzed by gas chromatography. The distribution of cell fatty acids in the strains is shown in Table 1 below, and anteiso-C ₁₅ : 0, iso-C ₁₅ : 0 and iso -C ₁₆ : 0 Fatty acid predominates.

fatty acid Amount (%) C _{14: 0} 0.4 C _{15: 0} 0.7 C _{16: 0} 1.3 iso-C _{14: 0} 8.2 iso-C _{15: 0} 20.3 iso-C _{16: 0} 14.3 C _{17: 0} 1.6 anteiso-C _{15: 0} 36.7 anteiso-C _{17: 0} 4.8 C _{16: 1} ω7c alcohol 4.0 C _{16: 1} ω11c 0.6 iso-C _{17: 1} ω10c 0.4 C _{15: 0} 2OH 1.3 iso-C _{14: 0} 3OH 1.1 iso-C _{15: 0} 3OH 1.6 iso-C _{16: 0} 3OH 1.2

The cell wall peptidoglycan of the isolated strain was analyzed by TLC, and the cell wall peptide glycan type of the strain was A1α (L-Lys diect). This is very distinguished from those in which Gram-positive bacillus cell wall peptideglycans with endospores generally contain meso-diaminopimelic acid.

In addition, the chromosomal diene of the strain was extracted and the G + C content was analyzed by liquid chromatography. The G + C content of the chromosome diene of the strain is 39 mol%, which is typical of strains classified as genus Bacillus.

After amplifying the bacterial 16S rRNA gene by PCR to obtain a large amount of 16S rDNA of BH724 strain, the nucleotide sequence of the 16S rDNA was analyzed [SEQ ID NO: 1], and the similarity of 16S rRNA with existing Bacillus bacteria. Was analyzed comparatively.

16S rDNA was amplified using two known universal primers. The base sequences of the two universal primers are as follows;

Forward primer: 27F ( E. coli 16S rRNA positions 8-27; 5'-AGAGTTTGATCCTGGCTCAG-3 ': SEQ ID NO: 2)

Reverse primers were 1492R ( E. coli 16S rRNA positions 1492-1510; 5′-GGTTACTTGTTACGACTT-3 ′: SEQ ID NO: 3).

The PCR reaction product was composed of 10 ㅧ reaction buffer, 2.5 mM MgCl ₂ , 50 mM dNTPs, 0.5 μl of 5U Taq polymerase (Bioneer co. Ltd., Korea), 20 pmol primer, and about 20-80 ng of purified DNA. The temperature conditions of each reaction for PCR were as follows;

For 40 seconds at 94 ℃ for DNA denaturation, 40 seconds at 55 ℃ for the annealing of the primer, and 1 minute at 72 ℃ for the synthesis of the DNA strand was used 28 times.

After completion of the reaction, the PCR product was electrophoresed in 1% agarose gel and stained with ethidium bromide, and the amplified PCR product was subjected to PCR purification kit (NucleoGen) for sequencing reaction. Purification was performed using the recommended method. The base sequence was determined using universal primers present in the gene. The sequencing reaction product was finally determined using an automatic base sequence analyzer (ABI prism 3100 genetic analyzer).

The comparison of the analysis of the flexible relationship between the existing Bacillus genus and 16S rRNA is shown in Table 2.

Comparison of similarity of 16S rRNA sequences between isolates and related Bacillus genus  Species Accession number Similarity (%) B. aquimaris JCM 11545 ^T AF483625 97.1% B. marisflavi JCM 11544 ^T AF483624 97.0% B. vietnamensis JCM 11124 ^T AB099708 97.0% B. shackletonii LMG 18435 ^T AJ250318 96.9% B. acidicola DSM 14745 ^T AF547209 96.6% B. sporothermodurans DSM 10599 ^T U49080 96.5% B. carboniphilus JCM 9731 ^T AB021182 96.5% B. flexus DSM 1321 ^T D78478 96.2% B. megaterium DSM 32 ^T X60629 96.1%

After investigating the morphology, physiological and biochemical characteristics of the strains, they were analyzed by the Burgy's manual of determinative bacteriology, the manual for the identification of medical bacteria (2nd ed.), And the base sequence of 16S rDNA. Strains were identified according to the phylogenetic tree.

Nucleotide sequence of 16S rDNA was analyzed and DNA-DNA hybridization (DNA-DNA hybridization) was performed on the standard strains showing the highest similarity with BH724T strain by phylogenetic tree.

Phylogenetic tree based on the nucleotide sequence of 16S rDNA is shown in FIG. BH724T strains were associated with B. marisflavi , B. aquimaris and B. vietnamensis .

As a result as above, the strain of the present invention is named Bacillus seohaeansis BH724T as a new strain and deposited on November 7, 2006 to the Korea Biotechnology Research Institute Gene Bank, an international microorganism depositing institution, accession number KCTC 11021BP. Was granted.

Example 2: Basophilic Alkaline Protease Production from Isolated Strains

Final selection of BH724T strains with better casein degradability than Bacillus subtilis KCTC 1028 strain, which contains 5% of opaque casein (skim milk) and 10% sodium chloride (NaCl) concentration After culturing at 30 ° C. for 24 hours in a controlled marine medium, the protease activity was measured using a supernatant obtained by centrifugation of the bacterial culture at 10,000 × g for 5 minutes, and casein was used as a substrate. 300 μl of supernatant and 1.2 ml of 0.6% casein solution dissolved in 100 mM Na-carbonate buffer (pH 10.0) were mixed and reacted at 37 ° C. for 15 minutes, followed by 600 μl of 10% trichloroacetic acid (TCA). The mixture was stopped and allowed to stand at 4 ° C. for 5 minutes to precipitate undigested casein, followed by centrifugation to obtain a supernatant liquid from which the precipitate was removed. In 500 μl of the supernatant, 1.5 ml of 7.5% Na ₂ CO ₃ and 1 ml of a dilution of a foline reagent were added and reacted at 30 ° C. for 30 minutes, and the absorbance of the reaction solution was measured at 660 nm. The activity of protease (pMSL1) was measured using a standard quantitative curve prepared by treating tyrosine in the same manner as described above. The absorbance of the mixture of coenzyme solution and casein for 1 minute at 37 ° C. produced 1 ㎍ of tyrosine. Change was defined as the enzyme active unit was used.

As shown in FIG. 5, the BH724T strain of the present invention showed the highest activity at 5% concentration and increased protease activity as the salt concentration increased, whereas the comparative strain decreased the enzyme activity as the salt concentration increased. Maintaining at least 70% of the highest activity, showing the optimum activity at pH 9.0 as shown in Figure 6 and maintaining at least 60% of the highest activity at pH 11, showing the highest activity at 40 ℃ as shown in Figure 7 50 It maintained more than 80% of the highest activity even at ℃.

Example 3: Comparison with Existing Bacillus

Comparing protease activity in the culture supernatant of the Bacillus strains patented as a protease producing strain and the culture supernatant protease activity of the BH724T strain showed excellent protease activity of the BH724T strain. Table 3 shows the protease activity in the bacterial culture supernatant using casein as a substrate.

division Enzyme Activity (unit / ml) Bacillus subtilis KCTC 1028 2,412 Bacillus licheniformis KCTC 3049 520 Bacillus seohaeanensis BH724T  6,512

Meanwhile, as shown in FIG. 5, the BH724T strain of the present invention exhibited the highest activity at 5% concentration as protease activity was increased with increasing salt concentration, in contrast to the decrease in enzyme activity with increasing salt concentration. It maintains high activity of 70% or more of the highest activity even at salt concentration of%. In the activity according to pH also showed the strongest activity at pH 7 ~ 9 as shown in Figure 6, the activity according to the temperature showed the strongest activity at 35 ~ 45 ℃ as shown in FIG.

Therefore, the productivity of the protease enzyme of the BH724T strain is not only superior to the existing protease producing strain, but also shows high activity under basophils and alkaline conditions, suggesting the industrial application of the protease produced by the BH724T strain.

As described above, Bacillus seohaeanesis BH724T (KCTC 11021BP), a new strain according to the present invention, can greatly improve the production rate of vial alkaline protease in the existing Bacillus bacteria and the enzyme industry for detergents. It is expected to be very useful for the food enzyme industry.

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Claims (1)

Bacillus seohaeanensis BH724T [KCTC 11021BP] to produce basophilic alkaline protease.

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