KR100220091B1 - Novel microorganism separated from korean nephila clavata, and produced protease - Google Patents

Abstract

The present invention relates to a novel microorganism isolated from the intestine of Nephila clavata of Korea and a protease produced therefrom, the novel microorganism of the present invention showing proteolytic activity Aranicola proteolyticus HY −3 is stable at a temperature of 20-40 ° C. and a pH of 6-10 and produces a protease having a molecular weight of about 51.5 kd.

Description

Novel Microorganisms Isolated from the Intestines of Korean Sugarless Spiders and Proteolytic Enzymes Produced therefrom

The present invention relates to a new enterobacteriaceae isolated from the intestine of Korean nephila clavata (Aranicola proteolyticus) HY-3 and the protease produced thereby.

Proteolytic enzymes are classified based on their specificity, and their industrial uses vary according to their characteristics. For example, there are endopeptidases that randomly cleave proteins suitable for the specificity of enzymes to form peptides, and exopeptidases that break down amino acid units along the peptide chain, depending on the acidity characteristics. It is classified into alkaline and neutral, and it is classified into heat resistance, mesophilic and high temperature according to temperature, and contains metalloprotase containing metal ions and serine residue in amino acid according to the structure of enzyme. It is also divided into serine proteases.

On the other hand, in the case of spiders belonging to insects, the characteristics of the regimen is well known to secrete a powerful toxic substance to kill prey to eat and then liquefy its body and consume it. Therefore, the present inventors believe that there are various enzyme systems capable of degrading foreign proteins in the intestines of spiders, and these enzyme systems are thought to be expressed by microorganisms living in spiders. The microorganisms showing proteolytic activity were searched. During the search for several spiders, microorganisms having excellent proteolytic ability from the intestine of Nephila clavata made in Korea, and in particular, have isolated microorganisms that exhibit significant proteolytic activity even at low temperatures, leading to the completion of the present invention.

It is an object of the present invention to provide a novel microorganism having excellent proteolytic activity and protease produced therefrom, isolated from the intestine of Korean sugarless spiders.

1 is an electron micrograph of Aranicola proteolyticus HY-3 isolated from the intestine of a Korean sugarless spider according to the present invention,

Figure 2 shows the taxonomic flexibility diagram of Aranicola proteoritis HY-3 of the present invention,

Figure 3 is the result of confirming the activity of the protease produced by the Aranicola proteoricicus HY-3 of the present invention on a neutral polyacrylamide gel.

Figure 4 shows the nucleotide sequence of the 16S rRNA of the Aranicola proteoricicus HY-3 of the present invention.

In accordance with the above object, the present invention provides Aranicola proteolyticus HY-3 isolated from the intestine of the Korean sugarless spider.

The present invention also provides proteolytic enzymes produced from Aranicola proteolyticus HY-3, which are stable at a temperature of 20-40 ° C. and a pH of 6-10 and have a molecular weight of about 51.5 kd. .

Hereinafter, the present invention will be described in detail.

The microorganism of the present invention can be separated from the intestine of Korean sugarless spiders as follows. First, the head of the sugarless spider is removed, and only the torso is soaked in 70% alcohol for 10 minutes to sterilize the surface. The stomach is excised and the intestine except for the body surface is placed in a homogenizer with physiological saline and homogenized. Then, the strain is formed on a solid medium to which 1% skim milk powder is added to form a transparent ring.

As described above, the microorganism of the present invention having proteolytic activity is a gram-negative bacterium that does not produce spores as a result of examining its strain characteristics, morphological, physiological and biochemical characteristics, and has a cell size of 0.3 to 0.5 μm. It is a rod-shaped cell having a cell length of 0.7 to 1.2 μm, and exhibits strong motility and similar morphological characteristics to enterobacteriaceae, β-galactosidase, urease and ornithine decarboxylase. It has the physiological and biochemical properties that produce citric acid, use citric acid, produce acid from alcoholic carbohydrates such as mannitol, inositol, sorbitol, reduce nitrate to nitrite, and show oxidase negative. The fatty acid composition in the cells was 16 saturated fatty acids (32%) and 17 cyclic saturated fatty acids (12%). And 16 and 15 carbon complexes (20%) were present as main fatty acids.

Based on the above morphological, physiological and microbiological characteristics, the microorganism of the present invention has been identified as a novel intestinal bacterium, which is not known at all until now, and it is impossible to identify the existing intestinal bacteria, thereby identifying the microorganism of the present invention. The ribosome subunit gene was isolated and analyzed for its nucleotide sequence, which showed 94-97% similarity to the existing intestinal bacteria. Based on the above morphological, physiological and biochemical characteristics and sequencing results, the microorganism of the present invention was named Aranicola proteolyticus HY-3, a novel intestinal bacterium.

According to the present invention, the novel microorganism Aranicola proteoricicus HY-3, isolated from the intestine of the Korean shaman spider, was deposited with the accession number KCTC 0268BP dated August 1, 1996 to the Gene Bank in the Biotechnology Research Institute of the Korea Institute of Science and Technology.

In order to isolate the protease produced from the Aranicola proteoriticus HY-3 of the present invention exhibiting proteolytic activity as described above, the microorganism is first cultured in a culture medium (bacto tryptone 0.5%, yeast extract 0.5%, potassium chloride). 0.05%, magnesium sulfate 0.02%, pH 7.0), and incubated at 25 ° C. for 30 hours, the culture was centrifuged to obtain a supernatant, and the protein was concentrated to prepare some purified coenzyme. The crude enzyme solution was electrophoresed on a 10% neutral polyacrylamide gel to form a transparent ring immediately at room temperature when the proteolytic activity was examined on pure plate medium containing only skim milk powder to separate the bands and to confirm the enzyme active fraction. The eluted band was electrophoresed with 10% SDS-polyacrylamide gel to confirm the subunit of the protease. As a result, the protease isolated from the microorganism of the present invention was not composed of the subunit as a single band and its molecular weight was About 51.5 kd. The separated protease immediately creates a transparent ring even under refrigerated conditions at 4 ° C., and when it is left overnight, it decomposes all the skim milk powders of a plate medium of 10 cm in diameter. As described above, the protease produced by the microorganism of the present invention shows activity at low temperature and can be purified relatively easily.

Hereinafter, the present invention will be described in more detail based on the following examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

Example 1 Isolation and Identification of Microorganisms from Korean Sugarless Spiders

(Step 1) Separation of Microorganisms

Korean shaman spiders collected from Daejeon and Chungcheong, Korea were first removed from the head and then the body was immersed in 70% alcohol for 10 minutes to sterilize the surface. The stomach of the sterilized spider was dissected and the intestines except the body surface were taken and placed in a homogenizer with physiological saline and homogenized. The homogenate was spread on a solid medium to which 1% skim milk powder was added, and colonies forming transparent rings were selected to obtain strains showing proteolytic activity.

(Step 2) Characterization of Isolated Strains

The microbiological characteristics of the strain producing the protease isolated according to the present invention are as follows.

1) Morphological characteristics

After culturing the strain obtained in step 1 under optimal growth medium conditions, gram staining and spore staining were confirmed to be gram-negative bacteria that do not produce spores. As a result of observing the morphology with an electron microscope, as shown in Fig. 1, it was a rod having a cell size of 0.3 to 0.5 μm and a cell length of 0.7 to 1.2 μm and distributed throughout the body of the cell (peritrichous). It is a highly mobile bacterium containing three or more flagella, consistent with the morphological characteristics of typical intestinal bacteria that produce nitrate reductase that can reduce nitrate to nitrite.

2) Physiological and biochemical characteristics

The physiological and biochemical properties of β-galactosidase, urease and ornithine decarboxylase are produced as shown in Table 1 below, citric acid is used, and acid from alcoholic carbohydrates such as mannitol, inositol, sorbitol, etc. Resulting in reduction of nitrate to nitrite and oxidase negative. Such physiological and biochemical characteristics could not be regarded as general characteristics of intestinal bacteria.

Characteristics Responsive Motility, 36 ℃ + Catalase + Oxidase (Kovac's) - β-galactosidase + Arginine dehydrolase - Lysine decarboxylase - Ornithine decarboxylase + Phenylalanine Deaminase - Indole generation - Vogues-Proskauer reaction + Hydrogen sulfide generation - Urea Hydrolysis + Gelatin hydrolysis + Citrate + D-glucose, acid + Lactose Fermentation + Sucrose fermentation + D-mannitol fermentation + Myo-inositol fermentation + D-sorbitol fermentation + L-arabinose fermentation + Raffinose Fermentation + L-Rhamnose Fermentation - Maltose fermentation + Trehalose Fermentation + Cellobiose Fermentation - Erythritol fermentation - Melibiose Fermentation - D-Arabitol Fermentation - Glycerol Fermentation + Amigdaline Fermentation +

3) Cell Chemical Characteristics

As a result of analyzing the fatty acid composition, (G + C) content and morphology of isopronoid quinone in the cell, the cells were harvested as shown in Table 2.For the fatty acid in the cell, 32% saturated fatty acid and 16 carbon atoms 12% cyclic saturated fatty acids and 20% complex with 16 and 15 carbon atoms were present as main fatty acids.

Characteristics G + C content (%) 56.6 Isoprenoid quinone Q-8 Major fatty acids (%) C12iO 3.5 C14iO 5.0 C16iO 32.0 C17iO cyclo 12.0 C16il isoI / C14iO 3OH 10.0 C16il w7c / C15iO 2OH 21.5 C18il w7c / w9t / w12t 11.5 (w9c / w12t / w7c; w12t / w9t / w7c)

4) Nucleotide sequence of ribosomal subunit gene

The ribosomal subunit gene (16S rDNA) was isolated from the cells and then lanes (Lane, DJ, 16S / 23S rRNA Sequencing, Nucleic acid techniques in bacterial systematics, E. Stackebrandt and M. Goodfellow [eds.], Wiley, 115- 175 (1991)) was synthesized by PCR (chain polymerization) method using Taq DNA polymerase using primers (8F, 1492R) recommended.

Synthesized ribosomal subunit genes were isolated purely, cloned into pGEM-T vector (Promega), transformed into E. coli JM109 cells, strains containing blue clones were selected, and tagged tagioxy terminator cycle sequencing. The base sequence was determined using a kit (Tag Dye Deoxy Terminator Cycle Sequencing Kit, Applied Biosystem).

The ribosome subunit gene investigated was 1406 bp, and the results are shown in FIG. 4.

(Step 3) Identification of Microorganisms

The morphological, physiological, biochemical and cellochemical properties investigated in step 2 were impossible to accurately identify the microorganisms. Although it is classified into the genus Serratia or Rhanella belonging to the family Enterobacteriaceae family, the National Institutes of Health (NIH) analyzes the nucleotide sequence of the ribosome subunit gene for more accurate identification. As a result of comparing the data of the ribosome database (RDP, University of Illinois, USA), which collects only the GenBank and ribosomal subunit genes operated by the phylogenetic tree, the microbial strain of the present invention is an endosymbiont of aphid (Acyrthosiphon pisum). ) And the taxonomically closest to the newest microbe. Figure 2 shows the taxonomy of the microbial strain of the present invention.

Therefore, the microbial strain of the present invention is named as Aranicola proteolyticus HY-3, which means a bacterium that produces protease derived from spiders according to the scientific naming system, August 1, 1996. As of date, it was deposited with the Gene Bank within the Biotechnology Research Institute, affiliated with the Korea Institute of Science and Technology, and was given accession number KCTC 0268BP.

Example 2 Measurement of Production and Activity of Protease from Aranicola Proteoriticus HY-3

(Step 1) Production of Protease

The microorganism strains isolated and identified in Example 1 were incubated at 25 ° C. for 30 hours after adjusting the pH to 7.0 in an enzyme production medium (0.5% of bland tryptone, 0.5% of yeast extract, 0.05% of potassium chloride, 0.02% of magnesium sulfate). . After centrifugation of the culture solution, the supernatant was taken, and protein was concentrated using Amicon's Centricon-30 (Centricon-30) to obtain a crude enzyme solution. The proteolytic activity was examined aseptically on the plate medium containing the crude enzyme solution. As a result, a clear ring was formed immediately after 5 minutes. Figure 3 is the result of confirming the activity of the protease produced by Aranicola proteoricicus HY-3 on the neutral polyacrylamide gel. The part was eluted again and subjected to electrophoresis on a 10% SDS-polyacrylamide gel. As a result, it was confirmed that the molecular weight was not more than 30 kd, specifically about 51.5 kd. The separated protease immediately produced a transparent ring even under refrigerated conditions at 4 ° C., and when it was left overnight, all the skim milk powders of the plate medium of 10 cm in diameter were decomposed. In this way, the production of protease from the microbial culture medium of Example 1 could be confirmed relatively easily.

(Step 2) Determination of activity and stability of heat and pH of protease

In order to investigate the effect of temperature and pH on the activity and stability of the low-temperature protease, the activity change according to temperature and pH was investigated using the crude enzyme solution obtained in Step 1 of Example 2.

The activity of protease was measured by Brown and Schmidz's method (Braun, V. and Schmitz, G., Excretion of a protease by Serratia marcescens, Arch. Microbiol., 124, 55-61 (1980)).

That is, 1.2 g of azocasein was dissolved in 50 ml of 50 mM phosphate buffer (pH 7.5) to prepare a substrate solution. 600 μl of substrate solution and 100 μl of cell culture were mixed and reacted at 37 ° C. for 30 minutes. 600 µl of% trichloroacetic acid was added and allowed to stand at room temperature for 1 hour. 300 μl of 10 N sodium hydroxide was added to 600 μl of the supernatant obtained by centrifugation at 7000 rpm, and the absorbance was measured at 414 nm. The titer of the enzyme was 1 unit of the amount of enzyme when reacting with the substrate for 1 minute to release an amino acid corresponding to 1 μg of tyrosine.

First, in order to confirm the stability to heat, the treatment was performed for 2 hours at each temperature from 5 ℃ to 80 ℃ and the residual activity was measured according to the above method, the activity was high at 20 to 40 ℃ as shown in Table 3, It was confirmed that the activity rapidly decreased above 40 ° C. and almost no activity at 50 ° C. The optimum temperature was found to be 40 ° C.

Temperature (℃) 5 10 15 20 25 30 activation 15.3037 23.3922 24.9394 32.0670 31.0564 42.3122 Temperature (℃) 35 40 50 60 70 80 activation 50.4485 50.4485 5.6439 5.5365 5.3792 0

Next, to examine the pH dependence, buffers such as citric acid and phosphoric acid (pH 5), sodium phosphate (pH 6-7), tris-hydrochloric acid (pH 8-9), glycine-caustic soda (pH 10-11) After dissolving the substrate in to prepare a substrate solution, the reaction was added for 30 minutes at 37 ℃ by adding the coenzyme solution obtained in step 1, the activity is measured and the results are shown in Table 4 below. As seen here, a titer of at least 80% was maintained at a broad pH of 6-10.

pH 5 6 7 8 9 10 11 activation 6.9722 26.3769 37.4550 40.0604 37.3372 28.5297 9.1681

As described above, the novel microorganism Aranicola proteolyticus HY-3 of the present invention, isolated from the intestine of Korean sugarless spider, is stable at a temperature of 20 to 40 ° C. and a pH of 6 to 10 and is about 51.5. Proteolytic enzymes having a molecular weight of kd are produced.

Claims (2)

Aranicola proteolyticus HY-3, which produces proteolytic enzymes isolated from the intestine of Korean Nephila clavata (Accession No .: KCTC 0268BP) A protease produced by the aranicola proteoricicus HY-3 strain of claim 1, which is stable at a temperature of 20-40 ° C. and a pH of 6-10 and has a molecular weight of about 51.5 kd.

Images