KR100658193B1 - New lipase DNA encoding the lipase transformated E. coli method producing lipase by the E. coli and uses of the lipase - Google Patents

Abstract

The present invention relates to a novel cryogenic lipolytic activity clone (lipase) through a phenotype-based search for a metagenomic library constructed from deep-sea sediments up to 1,400 meters deep in the Southwest Pacific (3 ° 89 'south latitude, 152 ° 49' long east). ) And analysis of the gene encoding this lipase was performed. The amino acid sequence predicted from the determined nucleotide sequence of the gene was confirmed to be a novel lipase. In addition, after developing an expression recombinant plasmid for mass production of cryogenic lipase, the plasmid was transformed into Escherichia coli, and the lipase was produced from the recombinant strain for expression, and the lipase was purified and purified. As a result of reviewing the application and the like, the novel lipase showed optimum activity at pH 8 and 25 ° C, and the comparison of temperature stability showed higher stability in a wider temperature range than other low temperature lipases.

Metagenomic Library, Recombinant Strain EML1, Cryogenic Lipase

Description

New low temperature lipases, genes encoding them and transformed Escherichia coli, methods for mass production of lipases using the same and application of the produced lipases {New lipase, DNA encoding the lipase, transformated E. coli, method producing lipase by the E. coli and uses of the lipase}

Figure 1 shows the amino acid sequence of the novel low temperature lipase and the nucleotide sequence of the gene encoding it.

2 shows a schematic for the development of a recombinant plasmid (pEML1) for mass production of novel cryogenic lipases.

Figure 3 shows the recombinant C. E. coli BL21 (DE3) / pEML1 mass produced cryogenic lipase and pure purified cryogenic lipase.

4 is a graph showing the activation energy and inactivation energy of EML1 lipase.

5 is a graph showing the temperature stability of EML1 lipase.

6 is a graph of the activity against pH of EML1 lipase.

The present invention relates to novel cryogenic lipases and genes encoding them, transformed EML1 Escherichia coli producing novel cryogenic lipases, methods of culturing them to mass produce lipases and applications of the produced lipases.

Lipases (glycerol ester hydrolases) are enzymes that catalyze the hydrolysis or synthesis of a wide range of water-soluble or non-aqueous carboxylic acid esters or amides. Hydrolysis, alcoholic decomposition It is involved in a variety of biocatalytic reactions, such as acidolysis, esterification, and aminolysis, maintains activity in organic solvents as well as aqueous solutions, and is a separate aid to catalysis. It is one of the high industrially applicable enzymes because it does not require a cofactor, exhibits substrate specificity for various substrates, and has isomer selectivity that shows activity for only one of the optical isomers. To date, many kinds of animals, plants, and microorganisms have been known to produce lipases, and research on the biochemical properties of lipases and the acquisition of lipase genes has been carried out, especially food, detergents, fine chemicals, cosmetics, Research into using lipases is being actively conducted in all industries such as environmental materials industry.

In the field of fine chemistry that synthesizes high value-added leading substances including pharmaceuticals, the synthesis of ester compounds by conventional chemical methods requires a lot of side reactions that consume energy and adversely affect the quality because they are synthesized at high temperatures and pressures. Not only does this occur, there is a difficulty in producing high-purity fine chemicals due to low conversion and purity of specific optical isomers. In recent years, in order to supplement these problems, the reaction using a lipase showing position specificity and optical activity specificity as a biocatalyst has been actively studied, but the application of the lipase has been limited due to the disadvantage of low activity at low temperatures.

In addition, lipase has been studied as an additive for detergents and bleaching agents because it has a function of facilitating the action of a surfactant by hydrolyzing fatty components with fatty acids or glycerol, which are well soluble in water, but the results of use are still poor. This is because of the disadvantage of poor activity of the lipase at low cleaning temperatures, poor or incomplete removal of fats or fats and oils.

The present invention has been made to solve such a conventional problem, to provide a novel low-temperature lipase that can be applied to produce high value-added fine chemicals with high purity of the optical isomer by maintaining high activity even at low temperatures. To do this. In addition, it is to provide a new low-temperature lipase suitable for industrial applications such as detergent additives or cosmetics by maintaining the activity even at low cleaning temperature.

The present invention to solve the above problems, by securing the gene of low temperature lipase to confirm the novelty through the determination of the base, a method for producing a large amount of lipase by transforming and culturing Escherichia coli by developing a recombinant plasmid for mass production The lipase was purified and purified to examine the characteristics and temperature stability of the enzyme.

That is, the present invention relates to a novel low temperature lipase, a gene encoding the same, and a transformed EML1 Escherichia coli producing a new low temperature lipase, a lipase mass production method using the same, and an application of the produced lipase. The present invention will be described in detail.

However, the present invention is not limited to the following examples.

Example 1 Gene Isolation and Microorganisms

The gene for producing a novel lipase of the present invention extracts environmental DNA from sediment up to 1,400 meters deep in the Southwest Pacific Ocean, and is purified to construct a library using a Fosmid vector. The average insert DNA size of the library was about 32 kb, with 8,000 clones constructed, corresponding to about 280 mb base pairs.

From the constructed metagenome library, isolated from clones showing activity in Tricaprylin plate medium, the vector containing this gene was named pEML1 and its host E. coli as EML1, respectively. As of January 21, it was deposited with Gene Bank, a Korean patent bacterial cycle deposit institution, and was given accession number KCTC10771BP.

Example 2 Cloning and Amino Acid Sequencing of Lipase Genes

After obtaining a large amount of fosmid DNA of ES01029D12 clone with tricaprylnin-degrading activity found from the metagenome library, it was cut to 2-4 kb using Nebulizer and then end-repaired. Each DNA fragment is blunt-ended using an enzyme to prepare a small size of DNA to be inserted. The pBluescript SK vector was cut with the restriction enzyme EcoR V, treated with calf intestinal phosphatase, and then ligation with the DNA to be inserted was transformed into E. coli DH5α. .

This was used as a tricaprylin plate medium with ampicillin (1% tryptone, 0.5% yeast extract, 1% sodium chloride (NaCl), 1% tricaprylin )) And colonies were formed by incubating at 37 ° C. for 24 hours to decompose tricaprylnin to form a transparent ring. As a result of culturing the plasmid to isolate it, a 7.6 kb sized plasmid containing 4.6 kb sized DNA was identified and named as pBEML1. Sequence analysis of 4.6 kb insertion DNA of pBEML1 was performed by designing a sequence kit, a T7 primer, a T3 primer, and a primer for sequencing. It was determined by Sanger dideoxy chain termination method. The assembly was performed using a vector enti (NTI) program, and four open reading frames (ORFs) were found using NCBI's ORF finder. Among them, OH lipase (ORF) consisting of 915 nucleotides was found. Predicting the encoded amino acid sequence based on the nucleotide sequence of ORF (ORF), it was identified as a lipase having a molecular weight of 33,600 consisting of 304 amino acids.

The lipase was most similar to the lipase produced by Bacillus thuringiensis as compared with the amino acid sequence in NCBI GenBank. However, the homology of 33% was shown to be a novel lipase.

Example 3 Development of Recombinant Plasmid for Mass Production of Lipase

Recombinant plasmids for mass production were developed as shown in FIG. 2 for mass production of cryogenic lipase produced by pBEML1 produced from the metagenome library. PBEML1 plasmid DNA containing lipase gene as template DNA and synthesized two primers; Forward primer (5`-GGA TCC ATG ACT TCA ACG GCA AA-3`) Reverse primer (5`-TGG Nucleic acid amplification reaction (PCR) was performed using AAT TTG CTC ATA AGG AA GCT T-3 ′). Both ends of the DNA were synthesized to have the recognition sites of restriction enzyme Bam HI and Hind III, followed by ligation with mass production vector pET-28a (+) treated with the same restriction enzyme. By ligation, the recombinant plasmid pEML1 for mass production was developed.

Recombinant plasmid pEML1 has a strong T7 promotor and translational signal inherent, enabling the production of desired lipases when E. coli BL21 (DE3), which contains the T7 RNA polymerase gene, is used as a host. .

The activity of the lipase was measured by a pH stat method in which olive oil was hydrolyzed by enzymes at a constant pH to titrate the amount of fatty acids produced with sodium hydroxide (NaOH), and one unit of enzyme was 1 per minute. It was defined as the amount of enzyme that produced μmol of fatty acid.

Example 4 Expression and Production of Lipase in Escherichia Coli

E. coli BL21 (DE3) with recombinant plasmid (pEML1) was transferred to a liquid medium (LB) containing 50 micrograms (ug) per ml of kanamycin (1% tryptone, 0.5%). After incubation in yeast extract, 1% sodium chloride (NaCl) until the absorbance audio (OD ₆₀₀ nm) reaches 0.6, IPTG was added to a final concentration of 1 mM at 25 ° C. After 12 hours of incubation, a rapase enzyme of 80 mg / 1 liter culture was obtained.

Example 5 Separation and Purification Method of Lipase

The lipase produced by E. coli BL21 (DE3) was purely separated as follows: The mass produced cells were recovered by centrifugation. The recovered cells were broken by ultrasonic grinding and centrifuged (12,000 × g, Only the supernatant obtained at 4 ° C. for 10 minutes was used as a crude enzyme solution.His Bind columns were equilibrated with binding buffer (500 mM NaCl, 20 mM Tris-HCl, 40 mM imidazole, pH 7.9), and then The obtained crude enzyme solution is passed through the column, washed with washing buffer (500 mM NaCl, 20 mM Tris-HCl, 60 mM imidazole, pH 7.9), and the imidazole concentration is increased to 100 mM under the same buffer conditions. The lipase was purified The specific activity of the purely isolated new lipase for olive oil was 99.2 units / mg, expressing the recombinant strain E. coli BL21 (DE3) / pEML1 and mass-producing cryogenic lipase and pure purified low temperature. Sex lipase is shown in Figure 3 The.

Example 6 Characteristics of New Lipohydrolase

The properties of the pure lipase separated from each other were investigated according to the following experimental example, and the following results were obtained.

According to Experimental Example 1, the results of measuring the optimum reaction temperature and thermal stability of the purely separated lipase are shown in Figures 4 and 5. As shown in FIG. 4, the novel lipase showed optimal activity at 25 ° C., the enzyme activity was sharply reduced above 40 ° C., and the activation energy was 3.28 kcal / mol as shown in FIG. 4. These properties confirmed that the lipase had low temperature properties.

Results for measuring the thermal stability of the lipase are shown in FIG. 5. That is, from 10 to 35 ℃ to maintain the activity of more than 80%, 40 ℃ or more than 65 degrees of activity was maintained at 40% or more, it was confirmed that the low temperature and high thermal stability.

According to Experimental Example 2, the results of measuring the stability of the lipase enzyme and the optimum pH of the lipase enzyme are shown in FIG. 6. As shown, the optimum pH was found to be 8 and stable over a wide range from pH 5 to 10.

According to Experiment 3, the results of measuring the effects of various metal ions and surfactants on the novel lipase enzyme activity are shown in Table 1 and Table 2. That is, the activity was sharply inhibited in calcium ions, but 1.5-fold increased in zinc ions. Although the surfactant was reversibly inhibited by sodium zodide sulfide (SDS), the activity was increased by surfactants such as Triton X-100 and Tween series.

TABLE 1 Relative hydrolysis capacity for metal ions for novel lipases

Reagnets Relative activity (%)  None 100 MgCl ₂  94 MgSO ₄ 113  CoSO4  97 Co (NO ₃ ) ₂ 108 CuSO ₄ 107 CaCl ₂  35 MnCl ₂  85 NiSO ₄  71 ZnSO ₄ 152 FeSO ₄ 109  EDTA  61

TABLE 2 Relative hydrolysis capacity for surfactants for novel lipases

Detergent Relative activity (%)  None 100  SDS  19  Triton X-100 121  Tween 20  81  Tween 40  84  Tween 60 115  Tween 80 142

According to Experimental Example 4, the results of the decomposition experiments of triglycerides and natural fats and natural fats and fats using the new lipase are shown in Table 3. In other words, the new lipases hydrolyzed tricaprylin, trilaurin, and trimyristin well, and natural fats, such as coconut oil, palm oil and tallow, are good at hydrolyzing solid fats. Appeared.

Table 3 Relative hydrolysis capacity of triglycerols and novel lipases for natural fats and natural oils

Natural Fats and Natural Oils Relative hydrolysis power (%) Olive oil 100 Cotton seed oil 68 Soybean oil 54 Peanut oil 82 Linseed oil 71 Corn oil 68 Malt (wheat germ oil) 93 Coconut oil 203 Palm oil 157 Uji (beef tallow) 125 Triglycerol Relative hydrolysis power (%) Triacetin 5 Tributyrin 11 Tricaproin 26 Tricaprylin 79 Tricaprin 72 Trilaurin 100 Trimyristin 98 Tripalmitin 65 Tristearin 14 Triolein 50

The novel low temperature lipases having the above properties are suitable for use as ingredients for detergents, cleaning and bleaching agents.

That is, the novel low temperature lipases according to the present invention may be used in the preparation of detergents, washing and bleaching agents, for example, powder washing agents, in combination with lipases individually or in some cases, and also as detergents, cleaning proteases or additives. It can be mixed with commonly used amylase, lipase, pectinase, nuclease and oxidoreductase, and the like, and can be added in an amount of 0.1 to 3% by weight, such as the amount of enzyme for other detergents.

In addition, the present invention may contain a general amount of detergent-containing ingredients, such as surfactants, bleaches and supports, and universal adjuvants necessary for the formulation.

Of the above auxiliaries, reinforcing agents, enzyme stabilizers, pollutant carriers, complexing or chelating substances, foam control agents and visual sharpening agents, fragrances, preservatives, antistatic agents, colorants, antibacterial agents, bleach promoters and peroxide bleaches Precursors; and the like.

In addition, the novel low-temperature lipase of the present invention for use as an additive in cosmetics, namely, the general cosmetic ingredients in the present technology, namely purified water, ethylenediaminetetraacetate acetate, allantoin, triethanolamine, dimethicone, carboxyvinyl polymer, carboxymethylcellulose, Glycerin, hyaluronic acid, enzyme stabilizers, DL panthenol, and the like can be used in combination in general amounts.

Experimental Example 1  : Thermal Stability and Optimum Reaction Temperature of New Lipase Enzyme

In order to determine the thermal stability and the optimum reaction temperature of the novel lipase enzyme, the experiment was carried out as follows.

The thermal stability of the enzyme solution was treated for 30 minutes in the enzyme stock solution at 5 ℃ interval in the temperature range of 10-65 ℃ and the residual activity was measured at the hydrogen ion concentration 8.0 using pH stat. Activity measurement was performed by adding olive oil as a substrate to a dispersion solution (0.5% arabic gum, 20 mM NaCl, 1 mM CaCl ₂ ) at 1% (v / v), adjusting the final volume to 20 ml, and emulsifying the enzyme solution ( 2 μl ≒ 140 μg) was added and the activity was measured by calculating the amount of alkali added to pH 8.0 using a pH stat.

The optimum reaction temperature of the purified enzyme was measured at pH 8.0 using a pH stat in the range of 5-60 ° C at 5 ° C intervals.

Experimental Example 2  : PH Stability and Optimum Reaction pH Measurement of New Lipase Enzyme

In order to determine the pH stability and the optimum pH of the new lipase enzyme, the experiment was carried out as follows.

The pH stability of the new lipase enzyme was measured using a 100 mM sodium acetate, 100 mM potassium phosphate, 100 mM Tris-HCl, and 100 mM glycine solution for 30 minutes in a buffer prepared at pH 5-11. The residual activity of enzyme was measured by the method.

In addition, in order to determine the optimum reaction pH, olive oil was emulsified in a dispersion solution at 1% concentration, hydrogen hydroxide (1 N) was adjusted to 6.0-10, and then measured by pH stat method at 25 ° C.

Experimental Example 3  : Experimental Effect of Surfactant and Metal Ion on Novel Lipase Enzyme Activity

To investigate the effects of surfactants and metal ions on the activity of novel lipase enzymes, the following experiments were carried out.

The effect of surfactant on the enzyme activity was treated with SDS, Triton X-100, Tween 20, 40, 60, 80 at 1% concentration for 30 minutes and olive oil at the optimum activity condition (25 ℃, pH 8.0). The residual activity of the enzyme was measured by the pH stat method using the emulsion.

To investigate the effects of metal ions and inhibitors on enzyme activity, enzyme stocks were prepared using MgCl ₂ , MgSO ₄ , CoSO ₄ , Co (NO ₃ ) ₂ , CuSO ₄ , CaCl ₂ , MnCl ₂ , NiSO ₄ , ZnSO ₄ , FeSO ₄ After adjusting the metal ion and EDTA to 1 mM and reacting at room temperature for 30 minutes, the enzyme activity was measured under optimum conditions by pH stat method.

Experimental Example 4  : Degradation of Triglycerides and Natural Fats and Natural Fats and Fats Using Novel Lipase

Substrate specificity for the enzyme triglyceride was determined by pH stat method with different fatty acid chain lengths. Triacentin (C2), Tributyrin (C4), Tricaporin (C6), Tricaprylin (C8), Tricaprin (C10), Trilaurin (C12), Trimystin (C14), Tripalmitin (C16), Tristearin (C18), Triolein (C18: 1 ) Were each emulsified in a dispersion solution so as to have a concentration of 1%, and measured by pH stat method at 25 ° C.

Substrate specificity for natural oils is 1% of olive oil, cotton seed oil, soybeen oil, peanut oil, linseed oil, corn oil, wheetgerm oil, coconut oil, palm oil and beef tallow, respectively. In the same manner as in the method was measured by the pH stat method.

The present invention relates to a novel cryogenic lipase and a gene encoding the same, a transformed EML1 Escherichia coli producing a novel cryogenic lipase, a method for culturing the mass produced lipase, and an application of the produced lipase, as described above. New low-temperature lipase nonionic surfactants increase enzyme activity 1.5-fold, improve enzyme activity at low temperatures, have low temperature and stability at a wide range of temperatures, and have higher activity in unsaturated fatty acids than saturated fats. It can be usefully used in high value-added fine chemical industry and cosmetics and detergent industry.

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Claims (6)

A gene encoding the lipase having the nucleotide sequence set forth in SEQ ID NO: 1. Lipase encoded by the nucleotide sequence of SEQ ID NO: 1. delete PEML1 recombinant plasmid comprising the nucleotide sequence set forth in SEQ ID NO: 1. Microorganisms transformed by pEML1 recombinant plasmid. A method of mass-producing lipase by culturing the transformed microorganism of claim 5.

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