KR101202261B1 - Lipases EM2L7 Isolated from Deep Sea Sediment - Google Patents

Abstract

The present invention relates to a lipase and esterase enzyme isolated from a deep sea bed, and a method for preparing the same, comprising a deep sea sediment that reaches a depth of 1,400 meters in the southwestern Pacific (3 ° 89 'south latitude, 152 ° 49' long east) Provided are a novel lipase and esterase enzyme isolated from a coastal sediment and a metagenomic library constructed from a tidal flat sediment in Ganghwado, and genes encoding them and a method for producing lipase and esterase enzymes.

Description

Lipases separated from the deep sea seafloor ＥＭ２Ｌ７ {Lipases ＥＭ２Ｌ７ Isolated from Deep Sea Sediment}

The present invention relates to a novel lipase, a gene encoding the same, and a method for mass-producing the lipase. In addition, the present invention relates to a novel esterase, a gene encoding the same, and a method for mass-producing the esterase.

Lipase (glycerol ester hydrolases) refers to enzymes that catalyze the hydrolysis or synthesis of a wide range of water-soluble or non-aqueous carboxylic acid esters or amides. alcoholysis), acidolysis, esterification, aminolysis, etc., participates in various biological catalytic reactions, maintains activity not only in aqueous solution but also in organic solvents, and supports the catalytic reaction process separately. It is one of the enzymes with high industrial applicability because it does not require cofactor, exhibits substrate specificity for a variety of substrates, and has isomer selectivity that exhibits activity only for one of the optical isomers. Until now, it has been known that many kinds of animals, plants, and microorganisms produce lipase, and studies on the biochemical properties of lipase and research to secure the lipase gene are being conducted. Research to use lipase is being actively conducted across industries such as environmental materials industry.

In the field of fine chemistry that synthesizes leading substances with high added value, including pharmaceuticals, when ester compounds are synthesized by conventional chemical methods, since they are synthesized at high temperature and high pressure, energy is consumed and various side reactions that adversely affect quality In addition to this, the conversion rate and the purity of a specific optical isomer have been low, making it difficult to produce high-purity fine chemical products. In recent years, in order to compensate for these problems, reactions using lipases that exhibit site specificity and optical activity specificity as a biocatalyst have been actively studied, but the application possibilities are limited due to the disadvantage that the lipase is less active at low temperatures.

In addition, lipase has been studied as an additive for detergents and bleach because it has a function of facilitating the action of surfactants by hydrolyzing fat components into fatty acids or glycerol that are well soluble in water. This is because fat or oil and fat components are poorly or incompletely removed due to the disadvantage that the activity of the lipase decreases at a low cleaning temperature.

Esterase is an enzyme that breaks down ester bonds to make low-molecular fats and alcohols, and has activity only against glycerol esters having an acyl chain of 10 carbons or less. Until now, many kinds of esterases have been found in various organisms including plants, including bacteria, yeast, and higher organisms, and studies on biochemical properties and esterase genes themselves have been actively conducted. Esterase is a hydrolysis reaction of lipid, acidolysis reaction, transesterification, exchange of fatty acids between triglycerides, It is used for the synthesis of esters and the like, and is a very important enzyme industrially. In particular, it can be used in the detergent industry as it can decompose fat components in laundry stains into hydrophilic substances that can be easily removed, and it is an anti-aging agent for cheese production and baking such as aging and adding flavor of cheese, or margarine with a natural buttery flavor. It can be used in the food and beverage industry such as manufacturing, the waste treatment industry, and the pharmaceutical industry as a major component of a fire extinguishing agent. In addition, esterases have unique properties such as substrate specificity, optical activity specificity, and site specificity, and thus their use is attracting attention to develop various bioactive chiral drugs. The production of chiral compounds useful for the production of such optically active drugs is attracting attention as a task that has a very great influence on securing competitiveness of the pharmaceutical industry and promoting public health.

The above important reactions are sometimes carried out effectively at high temperatures and under organic solvents. In particular, beef tallow and palm oil, which are mainly used for the production of free fatty acids in the food industry, remain solid at normal enzymatic reaction temperatures, so a high-temperature reaction environment is required for enzymatic processing of these materials. do. Therefore, there is an urgent need for a heat-resistant esterase that is stable at high temperatures.

The present invention is to provide novel lipase and esterase enzymes.

The present invention is to provide a method for producing novel lipase and esterase enzymes.

The present invention provides a lipase enzyme and a method for preparing the same. In addition, the present invention provides an esterase enzyme and a method for preparing the same.

The present invention provides an isolated DNA sequence encoding a lipase enzyme and a recombinant vector containing the same. In addition, the present invention provides an isolated DNA sequence encoding an esterase enzyme and a recombinant vector containing the same.

The present invention is a novel lipase and esterase and a gene encoding the same, a transformed E. coli that produces a novel lipase and esterase, a method for mass-producing lipase and esterase by culturing the same, and the produced lipase and esterase As described above, as described above, the activity of new lipases and esterases is excellent, has stability in a wide range of temperatures, and can be usefully used in high value-added fine chemical industries, cosmetics, and detergent industries.

1 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 1 and SEQ ID NO: 2 (EM2L1).
2 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 4 (EM2L2).
3 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 6 (EM2L3).
4 shows the lipase nucleotide sequence and amino acid sequence of SEQ ID NO: 7 and SEQ ID NO: 8 (EM2L4).
5 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 9 and SEQ ID NO: 10 (EM2L6).
6 shows the lipase nucleotide sequence and amino acid sequence of SEQ ID NO: 11 and SEQ ID NO: 12 (EM2L7).
7 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 13 and SEQ ID NO: 14 (ATL1).
8 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 15 and SEQ ID NO: 16 (ATL3).
9 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 17 and SEQ ID NO: 18 (ATL5).
10 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 19 and SEQ ID NO: 20 (ATL6).
11 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 21 and SEQ ID NO: 22 (ATL7).
12 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 23 and SEQ ID NO: 24 (ATL11).
13 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 25 and SEQ ID NO: 26 (KTL1).
14 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 27 and SEQ ID NO: 28 (KTL3).
15 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 29 and SEQ ID NO: 30 (KTL4).
16 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 31 and SEQ ID NO: 32 (KTL7).
17 shows the esterase nucleotide sequence and amino acid sequence of SEQ ID NO: 33 and SEQ ID NO: 34 (KTL9).

The present invention provides a lipase enzyme and a method for preparing the same. In addition, the present invention provides an esterase enzyme and a method for preparing the same. The manufacturing method is not limited thereto, but is preferably a manufacturing method by a genetic engineering method.

In a first aspect, the present invention provides a nucleic acid sequence encoding a novel lipase enzyme having SEQ ID NO: 7 or 11 and nucleic acid sequences equivalent to the sequence. In addition, nucleic acid sequences encoding novel esterase enzymes having the sequence of SEQ ID NOs: 1, 3, 5, 9, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33 and the sequence Equivalent nucleic acid sequences are provided.

"Equivalent nucleic acid sequence" includes the codon degenerate sequence of the lipase enzyme and esterase sequence.

"Codon degenerate sequence" refers to a nucleic acid sequence that is different from the naturally occurring sequence, but encodes a polypeptide having the same sequence as the naturally occurring lipase and esterase enzymes disclosed in the present invention.

In a second aspect, the present invention provides a lipase enzyme having the amino acid sequence of SEQ ID NO: 8 or 12 and functional equivalents thereof. In addition, the present invention provides an esterase enzyme having an amino acid sequence of SEQ ID NOs: 2, 4, 6, 10, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 or 34 and functional equivalents thereof. to provide.

In the "functional equivalent" of the present invention, the lipase of SEQ ID NO: 8 or 12 and S of SEQ ID NO: 2, 4, 6, 10, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32 or 34 Any or all of the terase enzymes are substituted, or amino acid sequence variants in which some or all of the amino acids are deleted or added to maintain the enzyme function are included. The substitution of amino acids is preferably a conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn) ) And sulfur-containing amino acids (Cys, Met). The deletion of the amino acid is preferably located at a portion that is not directly involved in the activity of the lipase and esterase enzymes.

In a third aspect, the present invention provides a recombinant vector comprising an isolated DNA segment of SEQ ID NO: 7 or 11 encoding the lipase enzyme. In addition, a recombinant vector comprising a DNA segment of an isolated SEQ ID NO: 1, 3, 5, 9, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33 encoding the esterase enzyme Provides.

The term vector used in the present invention refers to a nucleic acid molecule capable of transporting by binding another nucleic acid thereto. The term expression vector includes a plasmid, cosmid, or phage capable of synthesizing a protein encoded by each recombinant gene carried by the vector. A preferred vector is a vector capable of self-replicating and expressing the nucleic acid to which it is bound.

In a fourth aspect of the present invention, a cell transformed with a recombinant vector comprising an isolated DNA segment of SEQ ID NO: 7 or 11 encoding a lipase enzyme is provided. In addition, a recombinant vector comprising a DNA segment of an isolated SEQ ID NO: 1, 3, 5, 9, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33 encoding the esterase enzyme Cells transformed with are provided.

The term'transformation' used in the present invention refers to a change in the genotype of a cell by absorption of foreign DNA or RNA into a cell.

Suitable transformed cells include, but are not limited to, prokaryotes, fungi, plant, animal cells, and the like. Most preferably, E. coli is used.

In a fifth aspect of the present invention, there is provided a method of producing a lipase using a cell transformed with a recombinant vector comprising an isolated DNA segment of SEQ ID NO: 7 or 11 encoding a lipase enzyme. In addition, a recombinant vector comprising a DNA segment of an isolated SEQ ID NO: 1, 3, 5, 9, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or 33 encoding the esterase enzyme It provides a method for producing an esterase enzyme using cells transformed with.

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the examples.

< Example 1> Isolation of genes and microorganisms

For the genes producing the novel lipase and esterase of the present invention, environmental DNA was extracted from sediments up to a depth of 1,400 meters in the Southwest Pacific Ocean, and a library was constructed using a Fosmid vector through a purification process. The average insert DNA size of the library was about 32 kb, and 8,000 clones were constructed, which corresponds to a base pair of about 280 mb. It was isolated from clones showing activity in Tricaprylin plate medium from the constructed metagenome library.

< Example 2> Lipase And Esterase Genetic Cloning And amino acid sequence determination

After obtaining a large amount of fosmid DNA of a clone with tricaprylnin degrading activity found from the metagenomic library, cut into 2-4 kb using a nebulizer, and end-repairing Each DNA fragment is blunt-end using enzymes to prepare small-sized DNA to be inserted. The pBluescript SK vector was cut with the restriction enzyme EcoRV, treated with calf intestinal phosphatase, and then ligated with the DNA to be inserted to transform into E. coli DH5α. Tricaprylin plate medium with ampicillin added (1% tryptone, 0.5% yeast extract, 1% sodium chloride (NaCl), 1% tricaprylin) )) and incubated at 37°C for 24 hours to find a colony that decomposes tricaprylnin to form a transparent ring. This was cultured to isolate and confirm the plasmid. For sequencing of the inserted DNA, a sequencing kit, T7 primer, T3 primer, and primer required for sequencing were designed and used. It was determined by the Sanger dideoxy chain termination method. The assembly was performed using a vector NTI program, and an open reading frame ( ORF ) was found using an ORF finder of NCBI.

As a result, EM2L4 and EM2L7 appeared as new lipases, and EM2L1, EM2L2, EM2L3, EM2L6, ATL1, ATL3, ATL5, ATL6, ATL7, ATL11, KTL1, KTL3, KTL4, KTL7 and KTL9 appeared as new esterases ( Table 1 ).

New Lipases and Esterases Clone
name Lipase/
Esterase
(bp) Manifestation
vector enzyme
site amino acid characteristic activation pH pH
range Temperature Temperature
range temperament EM2L1 780
(SEQ ID NO: 1) pET 24a(+) NdeI and XhoI 259
(SEQ ID NO: 2) 8 7-10 20 5?40 C6 Esterase EM2L2 1215
(SEQ ID NO:3) pET 24a(+) NdeI and XhoI 404
(SEQ ID NO: 4) 9 7-10 30 10?40 C6 Esterase EM2L3 900
(SEQ ID NO: 5) pET 24a(+) NdeI and XhoI 299
(SEQ ID NO: 6) 8.5 6.5?9.5 15 5?60 C6 Esterase EM2L4 993
(SEQ ID NO: 7) pET 24a(+) NdeI and XhoI 330
(SEQ ID NO: 8) Lipase EM2L6 807
(SEQ ID NO: 9) pET 24a(+) NdeI and XhoI 268
(SEQ ID NO: 10) Esterase EM2L7 834
(SEQ ID NO: 11) pET 24a(+) NdeI and XhoI 277
(SEQ ID NO: 12) Lipase ATL1 912
(SEQ ID NO: 13) pET 24d(+) NcoI and Not I 303
(SEQ ID NO: 14) 8 7.0-9.0 35 20-50 C4 Esterase ATL3 918
(SEQ ID NO: 15) pET 24a(+) NdeI and XhoI 305
(SEQ ID NO: 16) 8.5 6.5-10 20 5?30 C4 Esterase ATL5 1080
(SEQ ID NO: 17) pET 24a(+) NdeI and XhoI 359
(SEQ ID NO:18) Esterase ATL6 870
(SEQ ID NO: 19) pET 24a(+) NdeI and XhoI 289
(SEQ ID NO: 20) 8 6.5-10 40 20-50 C6 Esterase ATL7 1248
(SEQ ID NO:21) pET 24a(+) NdeI and XhoI 416
(SEQ ID NO:22) Esterase ATL11 939
(SEQ ID NO:23) pET 24a(+) NdeI and XhoI 312
(SEQ ID NO: 24) 8 7.5-8.5 35 25-40 C6 Esterase KTL1 1566
(SEQ ID NO: 25) pET 24a(+) NdeI and XhoI 521
(SEQ ID NO: 26) 8 6.5-10 30 5?45 C4 Esterase KTL3 1500
(SEQ ID NO: 27) pET 24a(+) NdeI and Not I 499
(SEQ ID NO:28) Esterase KTL4 1059
(SEQ ID NO: 29) pET 24a(+) NdeI and Not I 352
(SEQ ID NO: 30) 8.5 6.5-10 20 5?25 C6 Esterase KTL7 951
(SEQ ID NO:31) pET 24d(+) NcoI and Not I 316
(SEQ ID NO:32) 8 7?9 30 15?35 C6 Esterase KTL9 1119
(SEQ ID NO:33) pET 24a(+) NdeI and XhoI 372
(SEQ ID NO:34) 8 6.5-10 30 5?45 C6 Esterase

< Example 3> Lipase And Esterase Development of recombinant plasmid for mass production

In order to mass-produce novel lipases and esterases found from the metagenomic library, a recombinant plasmid for mass production was developed. Nucleic acid amplification reaction (PCR) was performed using DNA containing lipase and esterase genes as template DNA and two synthesized primers. Restriction enzymes, as shown in Table 1, cut both ends of the synthesized DNA to have respective recognition sites, and then ligated with a vector for mass production treated with the same restriction enzyme to develop recombinant plasmids for mass production ( Table 1 ).

The recombinant plasmid contains a strong T7 promoter and a translation signal, so if E. coli BL21 (DE3), which has a T7 RNA polymerase gene, is used as a host, mass production of the desired lipase is possible.

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

S. atmospheres activity of the p- nitrophenyl butyrate was measured by using;; (pNPEs p -nitrophenyl esters) as the substrate (p -nitrophenyl butyrate pNPB) or other p- nitro petil ester. The reaction mixture was composed of 0.01 ml of 10 mM substrate dissolved in acetonitrile, 0.04 ml of ethanol, and 0.95 ml of 50 mM Tris-HCl buffer (pH 8.0) containing an appropriate amount of enzyme. The enzymatic reaction was carried out at 35° C. for 3 minutes. The amount of p-nitrophenol released during the reaction was measured at 405 nm. One unit of enzyme was defined as the amount of enzyme that required 1 μmol of p-nitrophenol per minute at 35°C.

< Example 4> Lipase And Esterase In E. coli Expression and production

Liquid medium (LB) containing 50 micrograms (ug) per ml of kanamycin (E. coli BL21 (DE3)) carrying the recombinant plasmid (1% tryptone, 0.5% yeast extract ( yeast extract), 1% sodium chloride (NaCl)) until the absorbance Audi (OD600 nm) becomes 0.6, then IPTG was added to a final concentration of 1 mM, followed by incubation at 25°C for 12 hours, Rapase and esterase enzymes could be obtained.

< Example 5> Lipase And Esterase Separation and purification method

The lipase and esterase produced by E. coli BL21 (DE3) were purely separated as follows: The mass-produced cells were recovered through centrifugation. The recovered cells were broken by ultrasonic grinding and then centrifuged (centrifugation). 12,000×g, 4° C., 10 minutes) to obtain a supernatant solution as a coenzyme solution The His Bind column was prepared with a binding buffer (500 mM NaCl, 20 mM Tris-HCl, 40 mM imidazole, pH 7.9). After equilibration, the crude enzyme solution obtained above is passed through the column After washing with a washing buffer solution (500 mM NaCl, 20 mM Tris-HCl, 60 mM imidazole, pH 7.9), the concentration of imidazole is 100 under the same buffer conditions. The lipase and esterase were purified by increasing to mM.

Attach electronic file of sequence list

Claims (7)

A protein having the amino acid sequence of SEQ ID NO. Gene encoding the protein of claim 1. Lipase having the amino acid sequence of SEQ ID NO: 12. A lipase gene, characterized by encoding the lipase of claim 3. Recombinant vector comprising SEQ ID NO: 11. A cell transformed with the recombinant vector according to claim 5. A method for producing a lipase enzyme, comprising culturing the transformed cell according to claim 6 and separating the lipase enzyme from the cultured cell.

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