KR100462842B1 - Halotolerant Protease-Producing Halomonas marisflava - Google Patents

Abstract

The present invention relates to a new basophilic bacterium capable of producing salt-resistant proteases that exhibit activity even at high salt concentrations, and to a method for producing protease using the same. The strain of the present invention isolated from the fermented food of high concentration salt is a proliferative bacterium Halomonas marisflava LS1 (KCCM 10457) that grows at a salt concentration of 5-15%, and the protein degradation produced by the strain The enzyme has excellent flame resistance property that maintains 70% or more of activity compared to 0% of salt concentration even at 20% salt concentration.

Description

Halotolerant Protease-Producing Halomonas marisflava}

The present invention relates to a novel basophil microorganism Halomonas marisflava LS1 that produces protease that is maintained at high salt concentrations.

Proteolytic enzymes are widely used around the world, accounting for more than 60% of the entire industrial enzyme market, and basic proteolytic enzymes are used in various fields such as detergents, food, leather, pharmaceuticals, and textile industries. In particular, salt-resistant proteolytic enzymes, which are active at high salt concentrations, have a wide range of use, and are often produced by fermentation of basophilic microorganisms, thereby reducing contamination of various germs in the production process.

In Korea, salted fermented foods such as jangjang, salted fish, etc., which use a large amount of salt, have a significant portion of the national diet. In the fermentation and ripening process of the salted fermented foods, flame-resistant protease plays a very important role. Using salt-resistant proteolytic enzymes in the process of producing large amounts of fermented food has the advantage of significantly shortening the aging process of the product. However, commercially available salt tolerant proteases are very insignificant.

Flame- resistant proteases are mainly composed of Bacillus sp. ( J. Micriobiol. Biotechnol. , Vol . 11, No. 4, p558-563, 2001; Appl. Biochem. Biotechnol. , Vol . 38, p83-92, 1993) Halobacterium sp., A basophilic microorganism (Korean Journal of Agricultural Chemistry, Vol. 33, No. 4, p337-342, 1990; Biotechnol. Bioeng. , Vol . 55, No. 8, p471-479) and Halomonas genus ( Bacteria and Aspergillus sp. ( J. Ind. Microbiol. Biotechnol. , Vol . 26, p230-). ( Halomonas sp.) (Korean Journal of Agrochemicals, Vol. 35, No. 4, p237-241, 1992) 234, p. 2001) is known to produce fungi, but is insufficient for industrial application. That is, the salt-resistant proteolytic enzymes produced by the microorganisms have a disadvantage in that it is difficult to apply to salt fermentation in Korea that uses a salt of 15% or more due to a drastic decrease in enzyme activity at high salt concentration.

Therefore, the technical problem to be achieved by the present invention is to isolate from the salted fermented foods the basophilic bacteria that produce flame-resistant proteolytic enzymes that maintain high enzyme activity even at high salt concentrations. To this end, in the present invention, a bacterium producing a transparent ring was isolated from a solid medium containing salt and casein, and the enzyme fermentation conditions of the halomonas marisflava LS1, which was identified and separated, were examined.

1 is a diagram showing the optimum pH of the reaction of the protease produced by LS1.

Figure 2 is a diagram showing the optimum temperature of the proteolytic enzymes produced by LS1.

3 is a diagram showing the effect of salt concentration on the activity of the protease produced by LS1.

In order to solve the above problems, the present invention uses a halomonas marisflava LS1, which is a basophilic bacterium capable of producing a flameproof protease that maintains high enzyme activity even at high salt concentrations, and a saltproof protease using the same. Provide a way to produce.

Proteolytic enzymes produced by the halomonas marisflava LS1 of the present invention shows excellent flame resistance, which maintains more than 70% of the activity in the salt-free condition at a high salt concentration of 15% or more, so that protein degradation of soy sauce, miso, salted fish, etc. It provides a way to improve the manufacturing process of fermented food, which is the main source.

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

Halomonas Marisflava used in the present invention was isolated from the salted fermented food LS1 is ripe. This strain is well mixed with physiological saline solution (0.85% NaCl) and gradually diluted by diluting canary fish sauce in agar medium (3% skim milk powder, 10% NaCl, 1.5% agar) for proteinase separation at an appropriate concentration. After 2 to 3 days incubation at 30 ℃ was separated by a method of separating the bacteria forming the largest transparent casein degradation ring around the colony. The isolated strain was selected as LS1 as a single strain through several pure separation processes, and was identified as Halomonas marisflava by analyzing the sequencing of 16S rDNA. 30 December 2002 Deposited in the Center for Microbial Conservation (Accession No. KCCM 10457).

Halomonas marisflava, a basophilic marine bacterium, has recently been reported to the academic community ( Int. J. Syst. Evol. Microbiol. , Vol . 51, pp . 1171-1177, 2001). Although known to be absent, the halomonas marisflava LS1 isolated from the present invention was characterized by producing proteases that can degrade casein at 10% NaCl concentration and secreting them extracellularly.

The LS1 isolated from the present invention was added with minimal salt (0% glucose, 0.2% milk, 0.2% potassium monophosphate, 1.4% potassium diphosphate, 0.6% sodium citrate, 0.1% sodium sulfate, 0.02% magnesium sulfate, pH 7.0). After adding 25% and incubating for 48 hours at 30 ° C and 150 rpm, LS1 did not grow at all without salt condition and was the most viable at 10% salt concentration, and showed good growth characteristics at 5 ~ 15% condition. It was. 10% of salt was added to the minimum medium, and the pH of the medium was adjusted to 5-9. The same culture resulted in the most growth at pH 7, and the optimum culture temperature was 26 ~ 30 ℃.

The protease activity produced by LS1 was mixed with 1% casein solution and LS1 culture medium from which the cells were removed, and then reacted for a predetermined time. It was measured by the change in absorbance at 660 nm.

In the present invention, the carbon source that can be used for proteolytic enzyme fermentation using LS1 may include ribose, xylose, glucose, fructose, galactose, sugar, lactose, maltose, dextrin, and the like. The proteolytic enzyme activity measured after 48 hours shaking culture by adding 1% of various carbon sources to a minimum medium containing 10% salt was the best when using galactose as a carbon source. As the nitrogen source, organic or inorganic nitrogen sources such as corn steep liquor, yeast extract, beef extract, peptone, tryptone, soyton, casamino acid, yuan, sodium nitrate, ammonium nitrate and the like can be used. Proteolytic enzyme activity measured after 48 hours shaking culture by adding 0.2% of various nitrogen sources to a minimum medium containing 10% salt was effective using beef extract and peptone. In addition, the salt concentration suitable for enzyme production was 5-15%.

The LS1 isolated from the present invention was incubated for 48 hours in a minimal medium containing galactose and a nitrogen source as a carbon source, and the enzyme was obtained by examining the optimum pH of the crude enzyme solution obtained by removing the cells, using a buffer of pH 6-12. The optimum pH of degradation was weakly basic protease in the range of pH 8-9, and the optimum temperature was around 40 ℃. The enzyme activity was measured by adding 0-20% salt to the casein solution to measure the enzyme activity. As a result, the protease activity was maintained at 70% or more even at the salt concentration of 0% even at 20% salt concentration. Flameproof properties were shown.

The present invention will be described in more detail with reference to the following examples.

Example 1 Isolation and Growth Characteristics of Basophilic Bacteria

LS1 used in the present invention was isolated from fermented salted fermented food. This strain mixes canary fish and salt and mixes Canary fish sauce which is ripening after 4 months in physiological saline solution (0.85% NaCl) and dilutes them stepwise to agar medium for separating protease at appropriate concentration. 3%, NaCl 10%, agar 1.5%) and then incubated for 2 to 3 days at 30 ℃ was separated by a method of separating the bacteria forming the largest case of the transparent casein degradation ring around the colony.

To the separated LS1, add 0-25% salt to the minimum medium (1% glucose, 0.2% yuan, potassium monophosphate 1.4%, potassium diphosphate 0.6%, sodium citrate 0.1%, magnesium sulfate 0.02%, pH 7.0) After shaking at 150 rpm at 30 ° C. and incubating for 48 hours, the results of the growth were shown in Table 1.

<Table 1> Effect of Salt Concentration on Growth of LS1

Salt concentration (%) 0 2.5 5.0 10 15 20 25 Growth (A ₆₆₀ ) 0.002 0.235 0.638 1.005 0.330 0.138 0.046

10% of salt was added to the minimum medium, and the pH of the medium was adjusted to 5-9.

TABLE 2 Effect of pH on Growth of LS1

pH 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Growth (A ₆₆₀ ) 0.237 0.513 0.874 0.908 0.704 0.635 0.567

10% of salt was added to the minimum medium, and the pH of the medium was adjusted to 7.0. The results of the growth of the same culture at 15 to 40 ° C. were shown in Table 3 below.

<Table 3> Effect of temperature on growth of LS1

Temperature(??) 18 22 24 26 28 30 34 36 Growth (A ₆₆₀ ) 0.573 0.697 0.765 0.903 0.924 0.842 0.580 0.528

Example 2 Enzyme Production Using Separating Bacteria

LS1 is separated from glucose and contains 1% of ribose, xylose, arabinose, glucose, fructose, galactose, sugar, lactose, maltose, dextrin, etc. in a minimum medium containing 10% of salt except glucose. After 48 hours shaking culture in, the enzyme activity of the crude enzyme solution from which the cells were removed was shown in Table 4 as the relative activity.

Table 4 Production of enzymes according to carbon source

Growth (A ₆₆₀ ) Relative enzyme activity (%) Arabinos 1.307 3.9 Xylose 0.979 37.9 Ribose 1.402 32.0 glucose 1.09 30.1 fruit sugar 1.463 40.8 Galactose 1.462 100 Sugar 0.427 21.4 Lactose 0.851 9.7 Maltose 1.196 5.8 dextrin 1.073 6.8

Organic and inorganic nitrogen sources such as yeast extract, beef extract, peptone, tryptone, soyton, casamino acid, yuan, sodium nitrate, etc., as nitrogen sources in a minimum medium containing 1% galactose and 10% salt, respectively, are isolated. After adding 0.2% and shaking culture at 30 ° C. for 48 hours, the enzyme activity of the crude enzyme solution from which the cells were removed was measured and shown in Table 5 as relative activities.

Table 5 Production of enzymes according to nitrogen source

Growth (A ₆₆₀ ) Relative enzyme activity (%) yuan 1.44 21.6 Sodium nitrate 3.49 18.0 Yeast extract 9.14 35.1 Trypton 5.71 61.3 peptone 4.26 84.7 Soyton 2.79 55.0 Beef extract 3.30 100 Cassamino 1.29 48.6

Example 3 Optimal pH of LS1 Protease Reaction

LS1 is enzyme producing medium (1% galactose, 1% beef extract, 1.4% potassium monophosphate, 0.6% potassium diphosphate, 0.6% sodium citrate, 0.02% magnesium sulfate, 10% salt, pH 7.0, 121 ℃ x 15) Minute inoculation) and incubated for 48 hours at 150 rpm at 28 ℃ shaking cells were removed by centrifugation to prepare a crude enzyme solution as a supernatant. The crude enzyme solution was mixed with 1% casein solution and 0.2 M buffer (pH 5, acetate buffer; pH 6, 7, phosphate buffer; pH 8, 9, Tris buffer; pH 10-12, glycine-NaOH buffer) and then 25 ° C. Enzyme activity was measured at. The change in enzyme activity according to pH is shown in FIG. 1.

Example 4 Optimal Temperature of the Proteolytic Enzyme of LS1

The crude enzyme solution prepared in Example 3 was mixed with 1% casein solution and 0.2 M Tris buffer (pH 9.0), and the enzyme activity was measured at 25 to 50 ° C. The change in enzyme activity according to reaction temperature is shown in FIG. 2.

Example 5 Salt Resistance of Proteolytic Enzymes of LS1

The crude enzyme solution prepared in Example 3 was mixed with 1% casein solution and 0.2 M Tris buffer (pH 9.0), and then salt was further added to a concentration of 0-20%, and enzyme activity was measured at 40 ° C. Enzyme activity change according to salt concentration is shown in FIG. 3.

The strain disclosed in the present invention provides a flame resistant protease that exhibits activity even at high salt concentrations. In addition, the present invention makes it possible to industrially produce flame-resistant proteolytic enzymes by identifying the growth characteristics of the strain and the production conditions of the protease.

Claims (2)

Basophil bacterium Halomonas Marisflava LS1 (KCCM 10457) capable of producing flame resistant proteases. A method of culturing halomonas marisflava LS1 (KCCM 10457) in a fermentation broth, and producing a flameproof protease from the culture.