KR0145486B1 - Proteus vulgaris u80 and new lipase produced therefrom - Google Patents

Abstract

The present invention relates to a new strain Proteus vulgaris K80 and a novel lipase produced therefrom, and more particularly, to a solvent resistant to alkali and protease from which the new strain Proteus vulgaris K80 is produced. It relates to a novel lipase with high stability.

Description

New strain Proteus vulgaris K80 and novel lipases produced from it

1 is a graph showing the relative activity and stability according to the pH (a) and temperature (b) change for the novel lipase of the present invention,

2 is a TLC analysis of the degree of degradation of triolein by the novel lipase of the present invention.

3 is a graph showing the nucleotide sequence and amino acid sequence of the novel lipase of the present invention.

The present invention relates to the new strain Proteus vulgaris K80 and a novel lipase produced therefrom. More specifically, the present invention is resistant to alkali and protease produced by the new strain Proteus vulgaris K80 and is a solvent. It relates to a novel lipase with high stability.

Lipase is an enzyme that hydrolyzes triglycerides containing long-chain fatty acids to diglycerides, monoglycerides, glycerol and fatty acids, and is widely used in food, chemical and leather processing industries.

In addition, the lipase serves to facilitate the action of the surfactant by hydrolyzing the fatty component in the washing process into fatty acids or glycerol that is well soluble in water.

Therefore, a lot of research on detergent lipase has been conducted. As a result, Humicola lanuginosa lipase mass-produced in Aspergillus Oryzae in 1988 was used in the enzyme market under the name of Lipoase ^TM . It started to be used and is now used all over the world.

Since then, Pseudomonas alcaligens (European Patent No. 0334462), Bacillus pumilis (European Patent No. 91-16422), Pseudomonas plantarii (European Patent No. 468102A1) ) And Pseudomonas glumae (European Patent No. 407225A1) have been found to be suitable for detergents.

However, lipases used for detergents must be alkali-resistant and resistant to alkaline proteolytic enzymes used together as detergent enzymes. Is not big.

In addition, lipases are generally used as biocatalysts in the synthesis of high value-added lead substances including pharmaceuticals due to their location specificity and optical activity specificity.

For this reason, the use of lipase as a biocatalyst has recently exploded, but there is a problem of poor solvent stability.

Therefore, the present invention has completed the present invention as a result of research to find an enzyme having high alkali resistance and proteolytic enzyme resistance and high stability to the solvent as an enzyme existing in nature to solve the problems caused by the use of the conventional lipase. .

Accordingly, the present invention provides a novel Proteus vulgaris K80 strain, a novel lipase produced by the microorganism, a detergent composition containing the enzyme as an active ingredient, and a DNA having a gene sequence encoding the same. have.

Hereinafter, the present invention will be described in detail.

The present invention relates to a novel Proteus vulgaris K-80 strain (KCTC 8642P).

The present invention also relates to a DNA having the following nucleotide sequence encoding a novel lipase produced by the new strain.

The present invention also includes a novel lipase having a molecular weight of 31,298 and having the following amino acid sequence:

Referring to the present invention in more detail as follows.

The present invention relates to a novel microbial Proteus vulgaris K-80 strain isolated from soil (KCTC 8642P) and a novel enzyme lipase produced therefrom and a detergent composition using the same. It is isolated from the sample and its microbiological characteristics are as follows.

[Morphological characteristics]

The bacteria were found to be gram-negative by gram staining, and the size of the rod was 0.5 μm × 1.0 μm as observed by scanning electron microscopy. In addition, the bacterium had three pragellas and exhibited a swarming phenomenon on a solid medium.

Physiological features

The isolated microorganisms do not produce beta galactosidase and betaglucosidase, as shown in Table 1, and do not have arginine dehydrolase, lysine dicarboxylase, ornithine dicarboxylase and cytochrome oxidase activity. It has tryptophan deaminase, urease and catalase activity, and gelatin degrading ability. In addition, the bacterium has hydrogen sulfide and indole production capacity, and has the ability to reduce nitrate to nitrile, indicating the typical Proteus vulgaris.

[Glucose Usability Experiment]

The results of the fermentation / oxidation experiment for culturing the separated microorganisms with different types of sugars and for the production of acid or gas and the assimilation experiment for determining the growth of cells were shown in Table 2 below. The results showed that glucose and sucrose could be oxidized and assimilated en-acetylglucosamine, maltose, gluconate and malate. These results were in good agreement with the properties of Proteus vulgaris.

[Identification and Naming of Isolated Microorganisms]

Through the above morphological and physiological characteristics and sugar availability experiments, it was confirmed that the microorganism isolated in the present invention belongs to Proteus vulgaris, but since there is no report that the existing Proteus vulgaris strain produces lipase, This isolated strain belongs to Proteus vulgaris and is considered to be a new strain producing lipase, so it is named Proteus vulgaris K80. Deposited and assigned accession number KCTC 8642P.

The method for producing and isolating a novel lipase from the microorganism of the present invention as described above, and the characteristics of the enzyme and the gene and amino acid sequence encoding the enzyme will be described through the following examples and experimental examples. It is not limited to these.

Example 1 Production of New Lipase

PH of medium in which 1% Polypeptone, 0.5% Yeast Extract, 0.2% Beef Extract, 0.2% Glycerol, 0.3% Salt, 0.2% KHPO, 0.2% KHPO and 0.005% MgSO were dissolved to produce lipase. Was adjusted to 7.2, followed by autoclaving at 121 ° C. for 15 minutes, and then inoculated with 1% of a culture medium incubated with Proteus Bvlgari K80 (KCTC 8642P) strain of the present invention, which was incubated for 6 hours in the same medium composition. The cultivation of the lipase produced by bacteria was more than 3,000 units / l when the flask was incubated for 24 hours at ℃, resulting in 5,000 units / l.

Example 2

Characteristics of Novel Lipolytic Enzymes (lipases)

A precipitate obtained by adding 30 to 50% of ammonium sulfate to the culture medium of Proteus vulgaris K80 bacteria (KCTC 8642P) was treated with Triton X-100 to remove fat component, and then purified purely using CM-Sepharose CL-6B column. The properties of the lipase were examined according to the following experimental example.

Experimental Example 1

Determination of molecular weight and N-terminal amino acid sequence of enzyme

SDS-PAGE of the purified enzyme with the standard protein showed a molecular weight of about 32,000. Extracting the protein on the gel and analyzing the amino acid sequence of the N-terminal was shown in Table 3. The isolated enzyme is a novel enzyme with significantly different lipases and N-terminus produced by the same Gram-negative bacteria Pseudomonas.

Experimental Example 2

Characterization of Novel Lipase Enzyme

The effects of enzyme temperature and pH on the purely separated lipase were investigated by pH titration. In the case of temperature, the activity of lipase continued to increase up to 60 ° C, but the enzyme activity decreased rapidly above 60 ° C. On the other hand, it can be seen that the enzyme activity is maintained to some extent even at 20 ℃. In order to measure the thermal stability of the lipase, the enzyme activity was measured at 37 ° C. after 30 minutes at each temperature. The activity began to decrease from 45 ° C. and no activity was observed at 55 ° C.

As a result of measuring the lipase activity at different pHs, the optimum pH was found to be 10, and after remaining at various pHs for 1 hour, the residual activity of the lipase was determined to be stable until pH 11. Such results have now been found to be novel enzymes with superior results to lipases used as detergents. In addition, considering that the pH of the washing process using detergent is 9-10, it can be seen that the enzyme has sufficient alkali resistance for use in detergents.

The effects of various metal ions on the enzyme showed that most metal ions were not inhibited except for mercury ions. This means that the lipase, unlike other enzymes, maintains its activity without structural modification in the presence of metal ions. In addition, lipase activity was not significantly inhibited against phenylmethylsulfonyl fluoride, iodoacetamide, and ethylenediaminetetraacetic acid used as protease inhibitors.

On the other hand, this enzyme has been found to be significantly more stable against SDS than other existing enzymes, and the lipase activity is greatly increased upon addition of deoxyclate. Thus, the enzyme can be used in the chemical industry based on the fact that it is stable against various metal ions, inhibitors and surfactants.

Experimental Example 3

Triolein Degradation Using Novel Lipase

Experiment to decompose triolein using the lipase produced in Example 1 was carried out. 100 mM Tris (pH 9.0) buffer containing triolein (4%) was added to the reactor at 37 ° C., and 100 Units of new lipase was added, followed by reaction for 60 minutes while stirring. Samples were taken by time and analyzed by TLC (chloroform: acetone: acetic acid = 95: 4: 1). As shown in the attached drawing 2, after 5 minutes, 1, 2 (2, 3) -diolein and monoolein and Oleic acid was produced. This means that the enzyme has 1, 3-position specificity, and the enzyme having such position specificity has optical activity specificity, so it can be used as biocatalyst for various chemical reactions. Can be.

Experimental Example 4

Solvent Resistance of Novel Lipase

The new lipase was made into 16 units per unit volume (ml), and 10% and 20% solutions of ethanol, DMSO, acetonitrile, isopropanol and acetone were made. As a result of measuring the fat hydrolysis titer, it was found that the result was very stable in a solvent as shown in Table 5, and thus it could be used as a biocatalyst as in Experimental Example 3.

Comparison: Determination of enzyme titer without solvent

Experimental Example 5

Resistance test for protease of new lipase

After adjusting the new lipase to have 20 units per unit volume (ml), add a commercial detergent protein hydrolase, Savinase (Novo Nordisk) to 1 unit per unit volume (ml) After 60 minutes of reaction, the proteolytic enzyme was fully reacted and the residual titer was measured. As a result, 74% of the initial lipase titer remained, indicating that the protease was significantly resistant to proteolytic enzyme. When the lipase is used, the remaining titer is 50% or less, indicating that the novel lipase of the present invention is highly resistant to detergent protein hydrolase.

Example 3

Cloning and Amino Acid Sequencing of Lipase Genes

Chromosome DNA of Proteus vulgaris K80 (KCTC 8642P) was completely cut with Hind III, then cut with Hind III and phosphatase

Ligation with pUC9 treated with (phosphatase) (CIP) was followed by transformation into reactive E. coli JM83. This was spread on ampicillin-added TCN plates and incubated at 37 ° C for 24 hours to find a colony that decomposed TCN to form a clear zone.

The plasmid was isolated by culturing this to identify a 8.0 kb plasmid containing a 5.3 kb insert and named pKL. Back transformation of pKL to E. coli JM83 showed that 100% of the colonies produced showed lipase activity.

PKL was cut with various restriction enzymes to perform subcloning and found to have Xbal and EcoRI sites.

pKL and pUC9 were cut with Hind III and EcoRI, and then ligated and transformed into E. coli JM83 to obtain 5.2 kb pKL2. The 2.5 kb insert DNA in pKL2 was eluted on an agarose gel, then ligated with pUC19 cut with Hind III and Xbal and transformed to obtain 4.7 kb pKL3. For nucleotide sequencing (Nucleotide sequencing), 2.0 kb-sized insert DNA of pKL3 was transferred to pBluscript SK to finally obtain pKLB.

In order to sequence sequencing of 2.0 kb sized DNA of pKLB, 200bp base sequences at both ends were read using forward primer (-40) and reverse primer. The Erase a Base system was used to obtain deletion subclones of various sizes, and the entire base sequence of 2.0 kb DNA was determined using forward and reverse primers. Got it. A program called MacMolly3.5 was used to find an open reading frame (LAF) of rapase consisting of 861 nucleotides (287 AAs) and 20 lipases isolated from P. vulgaris K80 (KCTC 86429). It was confirmed that the N terminal amino acids match (Table 3).

The lipase was found to be the most similar to Group II lipase produced by the strains of Pseudomonas genus but with a completely new molecular weight of 31,298 which differs by more than 60% in amino acid sequence.

Example 4

Used as detergent for detergent of new lipase

The laundry test was performed by adding 2 g of casein, 1 g of Congo red and 1 g of olive oil to 100 ml of tap water, making a suspension using a sonicator, and adding 5 × 5 cm cotton cloth to it. What was dried at 30 degreeC was made into the cotton cloth for laundry test. The detergent was tested by preparing a commercially available enzyme cleaner (general Korean product) and a commercially available enzyme cleaner containing 10 units of the new lipase. The washing power is 0.1, 0.3, 05g of detergent prepared in 1L of water, 2 sheets of washing cotton cloth are added to it, and the mixture is slowly stirred for 20 minutes to measure the concentration of the extracted pigment at 400 nm using a spectrophotometer. Washing force was measured. As shown in Table 6, the washability of commercial enzymes and the inventive enzymes was much better in the detergents using the fat hydrolase of the present invention. It can be seen that the characteristics are excellent.

Claims (5)

Novel Proteus vulgaris K80 strain (KCTC 8642P). A novel lipase having a molecular weight of 31,298 and having the following amino acid sequence. DNA having the following nucleotide sequence encoding the novel lipase of claim 2. A detergent composition containing the novel lipase of claim 2 as an active ingredient. Biocatalyst containing the novel lipase of claim 2 as an active ingredient.