KR100860083B1 - Novel gene encoding lipase from tidal flat metagenome and a lipase protein encoded by it - Google Patents

Abstract

A protein encoded by a lipase gene derived from tidal flat metagenome is provided to show excellent lipase activity and be mass-produced by culturing a transformant transformed by the lipase gene. A lipase protein includes an amino acid sequence of SEQ ID : NO. 2. A nucleic acid molecule encodes the protein and includes a sequence of SEQ ID : NO. 1. A transformant is transformed by a recombinant expression vector including the nucleic acid, wherein the transformant is E. col deposited as a deposition no. KCTC 10985BP. A method for preparing the lipase protein comprises the steps of: (a) culturing the transformant; and (b) recovering the lipase protein from a culture material of the step(a).

Description

Novel lipase gene derived from mudflat metagenome and lipase protein encoded therefrom {NOVEL GENE ENCODING LIPASE FROM TIDAL FLAT METAGENOME AND A LIPASE PROTEIN ENCODED BY IT}

1 is a nucleotide and amino acid sequence of the lipase LipG of the present invention. Stop codons are marked with *.

2 is a result of comparing the homology between the amino acid sequence of the lipase LipG of the present invention and the amino acid sequence of the similar lipase. The amino acids marked with * indicate well preserved lipase specific catalytic triads and oxygenanion holes.

3 is a phylogenetic tree of the lipase LipG of the present invention and various lipases selected from the known lipase family.

4 is a schematic diagram of a recombinant vector pET22b (+)-LipG comprising a novel lipase gene derived from a tidal flat metagenomic according to the present invention.

5 is a result of confirming the expression and purification of the desired 34 kDa lipase in Escherichia coli transformed with a tidal metagenome-derived lipase gene by SDS-PAGE,

lane M is the molecular weight standard marker;

lane 1 is cell lysate of transformed Escherichia coli;

lane 2 is a fraction eluted after adding the transformed E. coli cell lysate to the Ni-NTA column; And

lane 3 represents the final pure purified lipase through the Ni-NTA column.

Figure 6 is a graph showing the activity and stability of the lipase according to the pH.

7 is a graph showing the activity and stability of lipase with temperature.

The present invention relates to a novel lipase, and more particularly, to a lipase gene and lipase protein encoded therefrom isolated from the metagenome of soil microorganisms in the tidal flat. Proteins encoded from the genes of the invention exhibit excellent lipase activity.

Lipase is an enzyme that hydrolyzes ester bonds of triglycerides and is known to be produced by many kinds of animals and plants and microorganisms, and research on biochemical properties and genes is being actively conducted. In particular, due to its unique properties such as high substrate specificity, optical activity specificity and position specificity, it is an important enzyme which is very useful in the detergent, food, chemical and pharmaceutical industries.

Efforts to discover novel enzymes including the lipases have been made mainly for microorganisms that can be cultured, and various microbial enzymes found therefrom have been industrially used. However, many recent studies of molecular microbial ecology have demonstrated that more than 99% of microorganisms in nature are not isolated and identified through traditional culture techniques performed in laboratories (Amann et al., Microbiol. Rev. 59: 143-169, 1995; Hugenholtz and Pace, Trends Biotechnol . 14: 190-197, 1996; Ward et al., Nature 345: 63-65, 1990). Therefore, new researches are underway to make a library using metagenome, a genome of all microorganisms extracted directly from the natural world without culture, and to discover many new genes that have not been used since then and secure useful products. .

Metagenome is defined as the genome of all microorganisms in nature. In general, metagenomic studies consist of separating metagenomes from natural samples without culturing microorganisms, and then preparing them into libraries and introducing them into cultivable Escherichia coli. This is a method for securing useful products from microorganisms that have not been cultured, but it is difficult to obtain information on the microorganism from which the gene is derived.

The University of Wisconsin, USA, for the first time, has successfully isolated large-scale metagenomes, cloned them into bacterial artificial chromosome (BAC) vectors to build a metagenomic library, and isolated a broad range of antibiotics and genes involved in their biosynthesis. Gillespie et al., Appl. Environ. Microbiol. 68: 4301-4306, 2002; Rondon et al., Appl. Environ. Microbiol. 66: 2541-2547, 2000. The Institute for Genomic Research (TIGR) team is also building a total microbial metagenomic library of marine microorganisms in BAC vectors to explore the genetic resources of microorganisms that are not cultured from marine ecosystems.

Thus, the present inventors have isolated a novel lipase gene from a metagenome library of tidal flats in which various microorganisms exist, confirming that the protein produced therefrom exhibits excellent lipase activity and completed the present invention.

It is a primary object of the present invention to provide novel lipase genes and novel lipase proteins encoded therefrom which exhibit excellent lipase activity.

Still another object of the present invention is to provide a recombinant vector comprising the gene and E. coli transformed with the recombinant vector.

Still another object of the present invention is to provide a method for preparing lipase protein comprising culturing E. coli and recovering lipase protein therefrom.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

In order to achieve the above object, in the present invention, a DNA is extracted from soil samples of the tidal flat and cloned into a fosmid vector to produce a metagenome library, and the library is converted into a tricaprylin emulsion. It was plated and cultured on the added nutrient solid medium. Subsequently, the recombinant plasmid containing the lipase gene is isolated by searching for colonies that produce transparent rings, and the sequencing and amino acid sequences of the lipase gene are inferred through total sequencing, and the physical and chemical properties thereof are identified. Novel proteins with good lipase activity unknown to were identified.

Accordingly, the present invention provides, as one embodiment, a novel lipase gene ( SEQ ID NO: 1 ) derived from a tidal flat metagenome. Specifically, the process of separating the lipase gene from the mud flat soil was performed as follows. After the metagenome was isolated from the tidal flat sample, the metagenomic library was prepared by transforming Escherichia coli with phosphmid. The library was smeared in a nutrient solid medium to which 1% tricaprylin emulsion was added to select a strain having excellent lipase activity among strains that degrade tricapryline to form a transparent ring, and Recombinant plasmids were isolated and named pFosLipG. The recombinant plasmid pFosLipG selected above was analyzed for all nucleotide sequences by shot-gun sequencing. As a result, pFosLipG consisted of a chimeric plasmid containing two gene fragments with different guanine / cytosine contents ( SEQ ID NOS: 3 and 4 ), which were assigned to GenBank, USA, on November 7, 2007. Registered as DQ458963 and DQ478880, respectively. In addition, a lipase gene region having a nucleotide sequence of 903 bp of SEQ ID NO: 1 was determined from the GenBank accession no. DQ458963 fragment ( SEQ ID NO: 3 ).

The guanine / cytosine ratio in the entire nucleotide sequence encoding the lipase of the present invention is 46.29%, the molecular weight is about 34 kDa, and the isoelectric point is pI 5.5. The lipase gene of SEQ ID NO: 1 was identified as a novel gene encoding lipase, showing a homology of about 30 to 39% with the previously reported lipase gene population by searching with a BLAST database.

However, due to the degeneracy of the codon or in view of the preferred codons in the organism to express the lipase gene, the lipase gene of the present invention does not change the amino acid sequence of the lipase protein expressed from the coding region. Various modifications may be made to the coding region within the region, and various modifications or modifications may be made within the range not affecting the expression of the gene even in portions other than the coding region, and such modified genes are also included in the scope of the present invention. Will be well understood. Accordingly, the present invention encompasses polynucleotides having a base sequence substantially identical to the lipase gene of SEQ ID NO: 1 and fragments of the gene. Polynucleotides having substantially the same nucleotide sequence mean those having sequence homology of at least 80%, preferably at least 90%, most preferably at least 95%.

In another aspect, the present invention provides a lipase protein having an amino acid sequence such as SEQ ID NO: 2 encoded by the gene.

In the nucleotide sequence of SEQ ID NO: 3, an open reading frame (ORF) corresponding to the 10019 site at nucleotide number 9117 is a lipase protein coding region, and the protein encoded thereby is named lipase LipG. Lipase LipG expressed from the lipase gene of the present invention is composed of 300 amino acids and has an amino acid sequence as shown in SEQ ID NO: 2 ( FIG. 1 ).

Lipases of the present invention exhibit the following properties: stable in the pH range from 6.0 to 11.0, optimal activity at pH 8.0; Activity up to 55 ° C. and optimal activity at 40 ° C .; Activity is increased by divalent metal ions such as calcium chloride or manganese chloride; Both the 1 and 2 positions of triacylglyceol exhibit nonspecific lipase activity that can hydrolyze.

In addition, the lipase of the present invention is a catalyst in the consensus sequence of 217th aspartic acid (Asp), 285th histidine (His), and glycine-X-serine-X-glycine (Gly-X-Ser-X-Gly). It consists of the nucleophilic 169 th serine residue and has a trifunctional catalytic activity, conserved in lipases. Most bacterial and fungal lipases have a histidine-glysine (Gly) sequence as an oxygenanion hole sequence, whereas the lipase gene of the present invention has an arginine and glycine (Arg97-Gly98) sequence. This is the characteristic sequence of fibrous fungal lipase. In addition, LipG-like lipase enzymes in Table 2 are genes that have recently been identified through whole genome sequencing, and enzymes with no protein properties identified. A phylogentic tree was constructed using 32 lipase amino acid sequences belonging to eight lipase families classified according to amino acid sequences and amino acid sequences of LipG. LipG and six similar lipase-related enzymes It did not belong to any known family and confirmed that it constitutes a new family that is not defined.

The present invention provides not only lipases having the amino acid sequence of SEQ ID NO: 2 but also variants or fragments of amino acids having different sequences by deletion, insertion, substitution or combination of amino acid residues within a range that does not affect the function of the protein. It is included in the scope of the present invention. Amino acid exchanges in proteins and peptides that do not alter the activity of the molecule as a whole are known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, or the like. Accordingly, the present invention encompasses polypeptides and fragments thereof having substantially the same amino acid sequence as said lipase protein, wherein substantially identical polypeptides comprise at least 80%, preferably at least 90% and most preferably at least 95% Means those with homology.

The lipase gene and protein or variants thereof or fragments thereof may be isolated from nature, including soil metagenome, synthesized according to known DNA or peptide synthesis methods, or prepared by recombinant methods based on DNA sequences. When using a recombinant technique, the nucleic acid encoding the LipG protein is inserted into a suitable expression vector known in the art, and the recombinant expression vector thus prepared is transformed into a host cell such as E. coli to culture the transformed host cell. By doing so, lipG Genes can be cloned in large quantities or LipG proteins can be mass produced. Thus, in another aspect, the present invention provides a method for preparing lipase protein comprising culturing the transformed host cell and recovering lipase protein therefrom.

In the production of the recombinant expression vector, expression control sequences such as promoters, terminators, enhancers, membrane targeting or secretion, depending on the type of host cell to produce the lipase gene or protein, The appropriate sequence and the like can be selected and combined in various ways depending on the purpose. In addition, the recombinant vector may include a sequence for facilitating purification of the expression, and in a specific embodiment of the present invention, a histidine-tag (His-tag) is placed at the C-terminus of Ni-NTA (Ni-NTA). , Ni-nitriloteiacetic acid) column was used to purify the expressed lipase.

As mentioned above, one or more nucleic acid bases may be mutated by substitutions, deletions, insertions, or combinations thereof, as long as the genes of the present invention encode proteins having equivalent activity, which are also included within the scope of the present invention. do. The above-described protein having an amino acid sequence of SEQ ID NO: 2 is preferably encoded by a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1 , but the present invention is not limited thereto, and may encode a protein of the present invention having the same amino acid sequence. As far as possible, it may be encoded by a nucleic acid molecule having a nucleotide sequence having another nucleotide sequence that is substantially the same as the nucleotide sequence of SEQ ID NO: 1 . The sequence of such nucleic acid molecules may be single or double stranded, and may be DNA molecules or RNA (mRNA) molecules.

As another aspect, the present invention provides a recombinant vector comprising the gene of SEQ ID NO: 1 useful for the preparation of a novel lipase protein according to the present invention as described above, and E. coli transformed with the recombinant vector.

Nucleic acid sequences encoding LipG proteins of the present invention can be isolated from nature or produced artificially through synthetic or genetic recombination methods. The nucleic acid sequence encoding the LipG protein of the invention can be operably linked to a recombinant vector capable of expressing it to provide a LipG protein.

As used herein, a "recombinant vector" refers to a gene construct that is capable of expressing a protein of interest or RNA of interest in a suitable host cell, and includes a gene construct comprising essential regulatory elements operably linked to express the gene insert. In the present invention, "operably linked" refers to a functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA so as to perform a general function. The promoter and the nucleic acid sequence encoding the protein or RNA can be operably linked to affect the expression of the encoding nucleic acid sequence.Operative linkage with recombinant vectors is prepared using genetic recombination techniques well known in the art. Site-specific DNA cleavage and ligation uses enzymes commonly known in the art, and the like.

Vectors of the invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers, and can be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The expression vector may also include a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, includes the origin of replication. By the way, in the case of a recombinant expression vector comprising a gene encoding a lipase according to the present invention, when the lipase protein is expressed in the host cell, the activity is shown, as described above, tricapryline in the culture medium of the host cell By adding a lipase substrate, such as, it is possible to select a transformed host cell without a selection marker.

The signal sequence includes a PhoA signal sequence and an OmpA signal sequence when the host is Escherichia spp., And an α-amylase signal sequence and a subtilisin signal sequence when the host is Bacillus. The signal sequence, the SUC2 signal sequence, and the like, when the host is an animal cell, may use an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, and the like, but are not limited thereto.

The recombinant expression vector according to the present invention is transformed into an appropriate host cell, for example, Escherichia coli or yeast cell, and then cultured in the transformed host cell, thereby replicating a large amount of DNA of the novel lipase gene according to the present invention or protein. Can be mass produced. Suitable culture methods, media conditions, and the like according to the type of host cell can be easily selected by those skilled in the art from known techniques known to those skilled in the art.

Preferably, the host cell is Escherichia coli, and in one specific embodiment of the present invention, a recombinant vector pET22b (+)-LipG comprising a gene encoding a new lipase is constructed (FIG. 4), and this is Escherichia coli BL21 (DE3). And the activity of the lipase expressed from the transformed strain, E. coli transformed only with the pET-22b (+) vector not containing the gene did not decompose tricaprylin at all. On the other hand, E. coli transformed with the lipase LipG of the present invention exhibited strong lipase activity by effectively degrading tricapryline. It was confirmed that the lipase gene isolated from the tidal flat metagenome was expressed in E. coli transformants and secreted into the medium, and exhibited excellent lipase activity. The transformed E. coli BL21 (DE3) / pET22b (+)-LipG was identified in 2006. On September 12, it was deposited with KCTC 10985 BP with Korea National Institute of Biotechnology and Gene Bank.

As described above, since the protein encoded from the mudflat metagenome-derived lipase gene ( SEQ ID NO: 1 ) of the present invention exhibits excellent lipase activity, the lipase protein is widely used as a biocatalyst in biomedical and fine chemical fields as well as in the conversion of fats and oils. Can be utilized. Therefore, as another aspect, the present invention provides a method for non-specifically breaking down ester bonds of triglycerides using the novel lipase according to the present invention.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are intended to illustrate the present invention in more detail, but the content of the present invention is not limited to the examples.

Example  One: Metagenome  Build a library

10 g of soil samples collected from the tidal flats of Saemangeum reclaimed land in Buan-gun, Jeollabuk-do (100 mM Trisma buffer solution (Tris-HCl, pH8.0), 100 mM EDTA (ethylenediaminetetraacetic acid) , Suspended in 100 mM sodium phosphate (pH 8.0), 1.5 M sodium chloride (NaCl), 1% (w / v) CTAB (hexadecyl trimethyl ammonium bromide), and then anionic surfactant (sodium dodecyl sulfate (SDS)) The final 2% (v / v) was added and reacted for 2 hours at 65 ° C. Thereafter, the supernatant obtained through centrifugation was separated by 30% (v / v) polyethylene glycol containing 1.6 M sodium chloride (NaCl) ( Polyethylene glycol was added in the same volume and mixed well, and the precipitated DNA was separated by centrifugation, suspended in TE bufer, and the same volume of phenol / chloroform / isoamyl alcohol (25: 24: 1) and chloroform / isoamyl alcohol (c The mixture was extracted twice by adding hloroform / isoamyl alcohol) (24: 1), and isopropanol was added to the supernatant obtained by centrifugation to recover the DNA. After dissolving, impurities were completely removed and electrophoresed using pulse-field gel electrophoresis (PFGE) and partially eluted with Gelase (Epicentre) to partially digest DNA fragments of about 23 to 48 kb. DNA fragments were constructed using the MetaControl genome library using the CopyControl fosmid library construction kit (Epicentre).

To check the quality of the library, the transformants were randomly selected and recombinant plasmids were extracted and subjected to restriction enzyme treatment. All of them had recombinant plasmids, and the average size of the inserted metagenome was 35 kb.

Example 2 Screening and Isolation of Recombinant Plasmids Having Lipase Activity

Tricapryline emulsion (1% (v / v) tricaprylin, 1 mM calcium chloride (CaCl ₂ ), 0.5% (v / v) to search for lipase genes from the metagenome library made in Example 1 Nutrient solid medium (1% (w / v) trypton, 0.5% (w / v) yeast extract, 0.5% (w / v) sodium chloride added with gum arabic) NaCl), 1.5% (w / v) agar (agar) and incubated at 37 ℃. Since lipase decomposes tricaprylin, it was selected colonies that decompose tricapryline to form a transparent ring. Recombinant plasmids were isolated from colonies with the highest tricapryline resolution and named pFosLipG.

Example 3: Nucleotide sequence analysis and lipase gene sequencing of recombinant plasmid having excellent lipase activity

Recombinant plasmid pFosLipG isolated from the metagenomic library was sequenced using shot-gun sequencing. Specifically, DNA fragments made by the physical method using pipetting from pFosLipG were subcloned into pUC118 (TaKaRa) vector and performed using an automatic base sequence analyzer (ABI 3730 DNA analyzer). As a result, pFosLipG was a chimeric plasmid in which two DNA fragments with different guanine / cytosine content were inserted between the original pCCIFOS vector, and these DNA fragments were 15,320 bp and 15,364 bp, respectively. The base sequences of these genes are the same as SEQ ID NO: 3 and SEQ ID NO: 4 , respectively, and they were registered as GenBank (accession numbers) DQ458963 and DQ478880, respectively, on November 7, 2006. The ORF finder of the National Center for Biotechnology Information (NCBI) was used to identify only open reading frames (ORFs) with e-values less than 1 xe ^-10 , and each transcription was carried out using BlastX. The function of the decoding frame was predicted. A total of 24 transcriptional deciphering frames were identified, and the transcriptional deciphering frame corresponding to 10019 site at nucleotide number 9117 in the nucleotide sequence of SEQ ID NO: 3 was the lipase protein coding region, and the gene was named lipase lipG ( Table 1 ). The lipase gene of the present invention is composed of 903 nucleotide sequences and has a guanin cytosine composition of 46.26%, and the lipase LipG expressed from this gene is a lipase protein composed of 300 amino acids and a molecular weight of about 34,430 Da ( FIG. 1 ). .

Table 1 . Analysis of open reading frame of pFosLipG

LipG showed the highest similarity with Rhodopirellula baltica SH1 using the BLAST database, and showed a low homology of 30-39% with the previously reported lipase gene population. It was identified as a novel gene encoding lipase. In addition, the lipase of the present invention is the 169 th serine residue (Ser) and 217 th aspartic acid (Asp) in the consensus sequence of glycine-X-serine-X-glycine (Gly-X-Ser-X-Gly), It had a specific catalytic triad preserved in a lipase consisting of the 285th histidine (His) ( FIG. 2 ). In addition, most lipases derived from bacteria and fungi have histidine-glysine sequences as oxygenanion hole sequences, whereas the lipase genes are fibrous fungal lipase specific. It had the sequence arginine-glycine (Arg97-Gly98) ( FIG. 2 ). Based on the results of FIG. 2, a phylogenetic tree was constructed using 32 lipase amino acid sequences belonging to 8 lipase families classified according to amino acid sequences and amino acid sequences of LipG. LipG enzymes similar to LipG in Table 2 and LipG of the present invention did not belong to any known family, demonstrating that they constitute a new family not previously defined. In addition, LipG-like lipase enzymes, such as those shown in Table 2 , are genes that have recently been identified through whole genome sequencing, enzymes for which protein properties have not been identified.

Table 2 . LipG-like proteins

Example 4 Preparation of Transformant

Cloning the lipG gene of SEQ ID NO: 1 in the restriction enzymes Nde I and Xho I sites of pET-22b (+) (Novagen) to prepare a recombinant plasmid containing the lipG gene capable of mass production of the novel lipase of the present invention E. coli BL21 (DE3) was transformed into E. coli BL21 (DE3) / pET22b (+)-LipG (FIG. 4).

Recombinant plasmid pFosLip detected from the metagenome library was used as template DNA, and polymerase chain reaction (PCR) was performed using primers of SEQ ID NOs: 5 and 6 synthesized.

SEQ ID NO : 5'-CCCATATGATAAAGAAAGAATTATACGAG-3 '

SEQ ID NO : 5'-CCCCTCGAGTGTGATCAGGTTGTTCCAGAT-3 '

lipG gene N-terminal primer and C-terminal primer are Nde I and An oligonucleotide having the restriction enzyme cleavage site of Xho I and set forth in SEQ ID NOs: 5 and 6 . Recombinant vector pET22b (+)-LipG has a strong T7 promoter and read signal inherent, and when using E. coli BL21 (DE3) containing T7 RNA polymerase as a host, mass production of lipase is possible. In addition, a tag encoding six histidines is formed at the C-terminus to facilitate the purification of the lipase.

DNA fragments amplified by polymerase chain reaction were completely cut with restriction enzymes Nde I and Xho I and then linked with expression vector pET-22b (+) treated with the same restriction enzyme and calf intestinal phosphatase. ) To prepare a recombinant plasmid pET22b (+)-LipG for lipase expression (FIG. 4). The recombinant plasmid pET22b (+)-LipG was subjected to electron shock electroporation with E. coli BL21 (DE3) to prepare a transformed strain. The transformant was named Escherichia coli BL21 (DE3) / pET22b (+)-LipG and was deposited on September 12, 2006 to the Korea Biotechnology Research Institute Gene Bank (Accession Number: KCTC 10985BP).

Example  5: in transformants Lipase  Expression and Purification

Escherichia coli BL21 (DE3) / pET22b (+)-LipG prepared in Example 4 was treated with liquid nutrient medium containing ampicillin (ampicillin, 100 µg / ml) (1% (w / v) trypton, 0.5%). (w / v) yeast extract, 0.5% (w / v) sodium chloride (NaCl), incubated at 600 nm until the absorbance was 0.6, followed by IPTG (isopropyl-β-D) at a final concentration of 1 mM -thiogalactopyranoside) was added to the culture and incubated for 12 hours. E. coli BL21 (DE3) / pET22b (+)-LipG obtained by centrifugation was suspended in binding buffer (50 mM Trisma buffer (Tris-HCl, pH 8.0), 500 mM sodium chloride (NaCl), 10 mM imidazole). Then, it was crushed by ultrasonic grinding. Only the supernatant recovered by centrifugation was added to a Ni-NTA (nitriloteiacetic acid) column, and the lipase was eluted using an imidazole concentration gradient. In order to confirm the purified lipase as described above, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed and stained with coomassie blue ( FIG. 5 ). As a result, it was found that the lipase of the present invention was effectively produced at a molecular weight of about 35 kDa after 12 hours of expression, which is quite close to the molecular weight inferred from the amino acid sequence of the lipase, so that this protein band is new. It was confirmed that it is a lipase. In addition, the lipase of the present invention was expressed in the water-soluble form, the inactivation of the purely separated lipase in the purification through the Ni-NTA column increased 4.6 times to 458.8 Umg ^-1 and showed a 61% recovery.

Example 6: Characterization of Lipase Isolated from Tidal Flat Metagenome

Enzyme activity, specificity for substrates with various carbon lengths, effects on various metal ions, and site specificity of lipase LipG purified in Example 5 were measured as follows.

The reaction solution for measuring enzyme activity was obtained by 10 μl of 10 mM para-nitrophenyl ester substrate, 40 μl of ethanol, 940 μl of 50 mM Trisma buffer (Tris-HCl, pH8.0), and purification. While adding lipase enzyme solution and reacting for 5 minutes, para-nitrophenol decomposed from the substrate was measured as an increase in absorbance at 405 nm. One unit (U) was defined as the amount of enzyme that could be produced by hydrolysis of 1 μmol para-nitrophenol per minute.

(1) Characteristics of lipases against temperature and pH

In order to investigate the effect of pH on lipase activity, the activity was measured in various pH buffers and showed the maximum activity at pH 8.0 ( FIG. 6A ), and the residual activity of lipase was measured after standing at various pHs for 60 minutes. The results were found to be stable over a wide range of pH 6.0-11.0 ( FIG. 6B ).

As a result of investigating the effect of temperature on lipase activity, it showed optimum activity at 40 ℃ ( Fig. 7A ), and after remaining for 60 minutes at each temperature to investigate the stability to heat, the remaining enzyme activity was measured up to 55 ℃. Its activity was maintained in the temperature range and appeared to be stable ( FIG. 7B ).

(2) Properties of Lipases for Various Carbon Lengths

To determine the specificity for substrates of varying carbon lengths, para-nitrophenyl acetate (C2), para-nitrophenyl propionate (p3), para-nitrophenyl butyrate (p-nitrophenyl butyrate (C4), para-nitrophenyl caproate (C6), para-nitrophenyl caprylate (C8), para-nitrophenyl caprate (p-nitrophenyl caprate, C10), p-nitrophenyl laurate (C12), para-nitrophenyl myristate (C14), para-nitrophenyl palmitate (C16), And lipase activity was compared in the same manner as the lipase activity measurement method described above except that para-nitrophenyl stearate (p-nitrophenyl stearate, C18) was used as a substrate.

Table 3 . Mechanical Parameters for Para-Nitrophenyl Ester Substrates

As shown in Table 3 , among the various substrates, k- _cat t / K _m is the highest in para-nitrophenyl palmitate (p-nitrophenyl palmitate, C16), indicating that it has the highest activity. It was about 30 times higher than p-nitrophenyl butyrate (C4). Thus, LipG, which degrades substrates with relatively long acyl chains better, is lipase and not esterase.

(3) Effect of Lipase on Metal Ions

The activity of the lipase on various metal ions at various concentrations as shown in Table 4 below was measured. As a result, it was confirmed that the activity increased by 150% by 10 mM calcium ion (Ca2 +) or 5 mM manganese ion (Mn2 +). Furthermore, the activity is reduced by 54% by 0.1 mM EDTA, indicating that divalent metal ions such as calcium ions and manganese ions are essential for the lipase activity of the present invention.

Table 4 . Enzyme Activity of Lipase on Metal Ions

(4) Location specificity of lipase

2,3-dimercapto-1-propanol tributylate (TBDMP) and the indicator 5,5-disolol-bis (2- to determine the location specificity of the lipase Dinitrobenzoic acid) (5,5-dithio-bis (2-dinitrobenzoic acid), DTNB) was used to determine whether 1,3-specific or nonspecific lipase. This is the principle that the free thiol group produced by hydrolysis of TBDMP by lipase reacts with DTNB, resulting in absorbance at 412 nm. After 180 minutes of reaction with LipG, the absorbance value at 412 nm was 1.124, which is close to the theoretical value of 1.25, which can be obtained when all the thiol groups of TBDMP are hydrolyzed. Therefore, it can be seen that LipG is a nonspecific lipase capable of hydrolyzing both positions 1 and 2 of triacylglyceol.

While the present invention has been described in detail by way of examples, those skilled in the art will understand that such specific techniques are merely preferred embodiments and thus are not intended to limit the scope of the present invention.

As described above, the protein encoded from the mud flat metagenomic-derived lipase gene of the present invention shows excellent lipase activity, and the protein can be mass produced by culturing the transformant transformed with the gene. The novel lipases according to the present invention have excellent lipase activity and have potential for various industrial applications.

<110> Korea Research Institute of Bioscience and Biotechnology <120> NOVEL GENE ENCODING LIPASE FROM TIDAL FLAT METAGENOME AND A          LIPASE PROTEIN ENCODED BT IT <130> PA9611-814 / KR <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 903 <212> DNA <213> nucleotides of LipG <400> 1 atgataaaga aagaattata cgagataaca tgggaaaaca tagctccgcc ctataaagac 60 tacgagtatt ttaccgggcg tgaaaattat ccattcgatt ttaatacaaa agaattcagc 120 atagcaaatg catggtggct gatcgaggcg gcgacacttg tatatgcgga agaggagtat 180 gcccgggtat tatttcaaaa ggcaggattt cctgaggtca gatattttac ggataaaagc 240 acccagtgct atgcagtaag caataaagac gtccttgttg tcgccttccg gggaacagag 300 agcagaaagc gaaaggataa agacgatttt cgtgatatcg ttgaagatgt caaggcggat 360 gccgacttcc ggctggtaga ttccgggaaa aaggggaaag tacataaggg gttcagcgat 420 gcacttgatg aggtctggca ggaattgcat agctatgtca aaggactgca gaatgagggc 480 cgggctctct ggataaccgg acatagtctc ggtgctgcaa tagccacact cgctgcgtat 540 cgatttgaaa atgttcaggg cctgtatacg tttgggtctc ccagagtggg ggatgaagat 600 tttgtaaagg acttccgcgt tcccgcatat cgttttgaaa ataataatga tattgtctgc 660 aaagttccgc cgccggcccc cggcttgtat gctcatgcag ggaaattgaa atatatcgac 720 agtgagggaa acattcatga cgatatcagc ccctgggagc ggtggacgga tgaggttaaa 780 gggaggttca acaatgccat gagcacactc ggccatggtt ttcaaggtct tgtacctgat 840 gcgatcaagg atcacgttcc tgtattgtat gcaatacata tctggaacaa cctgatcaca 900 tga 903 <210> 2 <211> 300 <212> PRT <213> amino acids of LipG <400> 2 Met Ile Lys Lys Glu Leu Tyr Glu Ile Thr Trp Glu Asn Ile Ala Pro   1 5 10 15 Pro Tyr Lys Asp Tyr Glu Tyr Phe Thr Gly Arg Glu Asn Tyr Pro Phe              20 25 30 Asp Phe Asn Thr Lys Glu Phe Ser Ile Ala Asn Ala Trp Trp Leu Ile          35 40 45 Glu Ala Ala Thr Leu Val Tyr Ala Glu Glu Glu Tyr Ala Arg Val Leu      50 55 60 Phe Gln Lys Ala Gly Phe Pro Glu Val Arg Tyr Phe Thr Asp Lys Ser  65 70 75 80 Thr Gln Cys Tyr Ala Val Ser Asn Lys Asp Val Leu Val Val Ala Phe                  85 90 95 Arg Gly Thr Glu Ser Arg Lys Arg Lys Asp Lys Asp Asp Phe Arg Asp             100 105 110 Ile Val Glu Asp Val Lys Ala Asp Ala Asp Phe Arg Leu Val Asp Ser         115 120 125 Gly Lys Lys Gly Lys Val His Lys Gly Phe Ser Asp Ala Leu Asp Glu     130 135 140 Val Trp Gln Glu Leu His Ser Tyr Val Lys Gly Leu Gln Asn Glu Gly 145 150 155 160 Arg Ala Leu Trp Ile Thr Gly His Ser Leu Gly Ala Ala Ile Ala Thr                 165 170 175 Leu Ala Ala Tyr Arg Phe Glu Asn Val Gln Gly Leu Tyr Thr Phe Gly             180 185 190 Ser Pro Arg Val Gly Asp Glu Asp Phe Val Lys Asp Phe Arg Val Pro         195 200 205 Ala Tyr Arg Phe Glu Asn Asn Asn Asp Ile Val Cys Lys Val Pro Pro     210 215 220 Pro Ala Pro Gly Leu Tyr Ala His Ala Gly Lys Leu Lys Tyr Ile Asp 225 230 235 240 Ser Glu Gly Asn Ile His Asp Asp Ile Ser Pro Trp Glu Arg Trp Thr                 245 250 255 Asp Glu Val Lys Gly Arg Phe Asn Asn Ala Met Ser Thr Leu Gly His             260 265 270 Gly Phe Gln Gly Leu Val Pro Asp Ala Ile Lys Asp His Val Pro Val         275 280 285 Leu Tyr Ala Ile His Ile Trp Asn Asn Leu Ile Thr     290 295 300 <210> 3 <211> 15320 <212> DNA <213> DNA sequence 1 of pFosLipG <400> 3 tgccatcaac atagaggtca gtattgataa cagcagtccc ggtaacgaag aagttgctgt 60 ccactgacag gtcacccgtc atggtatcgc ctgctgtgtc cacccagtta tctgtgcttg 120 tcgtcatgaa tgctgttgag tccagaccat ccagcaggtc agcatcaaga gttgatgcac 180 tcccgtcatt tgcaagcact gtaggcatta tctcactatc tctggctatc aatgcatcta 240 tccttgcttc aaatactgtg ccagagatta tatctgatcc agaatgagta tggattgccc 300 ctgcaaaatc aattgcatgc ttcccgtcaa ctgtgtctgc atcagtaaaa gtaataccgg 360 ttaatcccga tccatcacca gtgaaggagt tggccgtaag tgctccgtta acaataacaa 420 tgtcattgtc aaattcccca taaataagag gtgtgctggt attagagttg tctatgtaga 480 gcttgtttga acctgtttcg ttatatcctg tttggtagcc aataaagaca ttgcctgaac 540 cggttgtgtt gtaaccagcc ccatatccat taaatgtatt catgcttcca gctgtgtttg 600 agtatccggc accttgacca gtaattgtat tgtagcctcc agtagtattt tcatagccac 660 ttgaatgtcc aataaaagta ttgcccgctc cagaagtgtt acggttacca gagctcatac 720 cattaaaagt attatagctt cccgttgaat ttgatattcc tgaatatgta ccagtaaacg 780 tgttagctga accacttgaa ttagatgaac ctgctgagct cccgataaaa gtattataaa 840 agtttgtatt tgcgtaacct gctctatacc ccagaaaggt attgtcttca ccaacaacat 900 tgctccttcc tgcaaatgat ccgataaaag tatttaatgc acctatattg ttagataatc 960 ctgattcttt gccaataaaa gtgttagaaa atccggatgt attttcatag ccacttgaat 1020 gtccaataaa agtattgccc gctccagaag tgttacggtt accagagctc ataccattaa 1080 aagtattata gcttcccgtt gaatttgata ttcctgaata tgtaccagta aacgtgttag 1140 ctgaaccact tgaattagat gaacctgctg agctcccgat aaaagtatta taaaagtttg 1200 tatttgcgta acctgctcta taccccagaa aggtattgtc ttcaccaaca acattgctcc 1260 ttcctgcaaa tgatccgata aaagtattta atgcacctat attgttagat aatcctgatt 1320 ctttgccaat aaaagtgtta gaaaatccgg atgtattttc atagccactt gaatgtccaa 1380 taaaagtatt gcccgctcca gaagtgttac ggttaccaga gctcatacca ttaaaagtat 1440 tatagcttcc cgttgaattt gatattcctg aatatgtacc agtaaacgtg ttagctgaac 1500 cacttgaatt agatgaacct gctgagctcc cgataaaagt attataaaag tttgtatttg 1560 cgtaacctgc tctatacccc agaaaggtat tgtcttcacc aacaacattg ctccttcctg 1620 caaatgatcc gataaaagta tttaatgcac ccatattgtt agataatcct gattcggtgc 1680 caataaaagt gttataaaat ccagatgtat tgttaaatcc tgcatcactc ccaataaacg 1740 tattgtcatt tgccgtattg tttaatcctg ctctatagcc taataacgta ttatcgtttc 1800 cggttatatt ccactttccc gcacttctac ccagaaaggt attattgatt ccggtttcgt 1860 tggcataacc ggcacggtta ccaatgaaag tattgctttt ccccgttgta ttagcatatc 1920 cagctatgta tccaatgaac gttgaaaagt catcatctcc tgaaactaat cctgcactct 1980 gaccgtaggc agtgttgctg gctccctcca tacccgctaa tgcactgaca ggaatgagca 2040 cagctactcc gaaaagaaga cttatagcaa ccaacaataa tttcatttta ctaccccctt 2100 tatatgagtt ctcgaacatt atttttcaaa cataaaaaag ccgagcgtac ttatactgtc 2160 acctctggta gctcagccat ctcccctaca agacacgaac cattaggtaa actcatccat 2220 ccatatatgg tctatatcaa tattacaata gatctgaaaa acgtattgtt ataattacta 2280 cttggagaga attattataa ttctatcatt aaaactcaat acatcaaaac gcacttatag 2340 aagttataaa tatttcatat atgcacgaac tggcaaataa ggatttacat ttgcaatctt 2400 gtattttgag gaagagatag agttattgat aacttcaaca tattaatgaa tcatgagtgc 2460 tctcaagctt attgatatgt agcggtaatc agcaaaaaac atttaaaatc aatatatcat 2520 gtggttatca gtaatctgag atccatcaaa aactgaacat accatcatga tatcaatagg 2580 agagtcttaa ataaaactga taattgccgt ttgaaataaa cagtattaat cagagtcagc 2640 cattgtcaca attctgttga aactctctcc attggggata tcagtgttta ggtgggaagg 2700 gttagagatt tgatatactg taatcaaaca gtgtttttaa acattacttt tgagggaggg 2760 ataaatggct gcaacaacta aaaagaagag ccctgtatca aaaaccacaa ggaaaagaaa 2820 agtgaaaaag agcgctgttt ctttcggaga agttgagtca tccatggaca agttggctct 2880 tgatgacttg acgcatacta ctaaggaaga gcttaggaaa ctgggggata aaattcatga 2940 agcaacggat aaaggtgttc atatggttaa ggacatagct gatcatgtcc agcgttttgc 3000 caatgacgct acagagctta cgaaacttaa aatagagctc cataatctaa aaaacgaaag 3060 aacaaaacta tacactttaa tgggagagca gttacgaaac ctccatatct ccaaaaaatt 3120 gacaaacatt cagaaacggt tcaaagatga ttttaaacga ctcgatgagc ttgaaacaga 3180 aatatcagaa ataaagaaga aggctgccag aatccctatt gctgaggata taaaaaaact 3240 ctgattccta ttctgtcttc attttacata cgttaaaaaa aatcagcttt gaaagcaata 3300 ttttaaacat aaagcaaatt tgtccgtgga ttaaatctat tatatattgc acttaagttg 3360 gagcaggccg ggtcatttct ttcctgagac ttttctggca ctaaagactt agtctttagt 3420 ctgtttctcc ggagcgtcct gcatcttttc tccgatttcc tcaatttttt ctcctgccac 3480 ttccaccact tcctccaccg cctcccgtgt ctcttcggct gcatctttca tcatctcttt 3540 tgtctcttcc gcagcctcat ctatcttttt tcccgcacgc tcggccggac cttccttctc 3600 acatgctgca aacgttaaaa taagaagcaa actgcacgtc agtaagccta tattcctaaa 3660 gattgtcata tttcgctcct ttcagtcttt tttatcgtta aattattact taccgatttg 3720 acaccctttg ttgctttcgc aattcggagc agcagctcct ctgttttacc gctcggcata 3780 aaaccattca gtaagatatc tcctccaatg tctcaataca ggtgtttagt tagattttat 3840 cacaggttta taagattcgg gaggaagagt gacctactcc ccccatatct cgatacagat 3900 gttaagttag agaaggagat atggacatgg agagaaacac tacacagcag aatagatcac 3960 caagtatccc agagaatcaa agattctact tgggaagggt gatcgttggg cagataagga 4020 taaacgaatt aaagcttcaa tacattcaga agtgacagat tattgataac ttcaacttat 4080 cgataaccca ctgtgcacta aatgtgcacc tgacaggata aaaaaaggga taacaccgta 4140 tagatgctat cccttcagtt tacgtctttg caagtaatat caagttacgg caagcaaggt 4200 atagttccca ctactatatc ctacatgact cttaatcagc ttgtcgcagg ttcgatccct 4260 gcacggctca ccatttaaaa aaatcaagga atccggacac ttactatacc tggattcctt 4320 tttttatcct cttcataata tcttcatatt actctgatat cttaaggtca ggaataaacc 4380 ataagagggt gctggcactg ggtaaaagat caattcctgc ctgctaagag ccaaccccga 4440 agagtaacac ctcggggaag atcttttatt cagccatgtc ccctacattt gtcggcaccc 4500 tcttacatat gtaacctctg tgtactgtga tccgacttcc ggttgccgta acggaaacgg 4560 aaaacgggca tagcatttcg ggcatcggac aatggtctac ggatttcgga caacgggttt 4620 gatgtctgct tttttgcgga aagttagatg aagctcttgt atttgtccag gggcgggtct 4680 atgatctcaa cccccatggc attgatcagg ccgtggggag ggagttttga cgaccatctg 4740 atgatgcatg tgagttcaag ttgttcggca ggctggatct cgaactttaa ttttatctct 4800 ttgccgggtg gtaagctcag gtccttttcc gtgcttacga tcgtgaccct cgcaccgctt 4860 gcagagaggt tctcgactat cccctgaaag gtcctatctc cgtaagtgat ctctgcctta 4920 tagccggcgg ttattctttt ggcttgtctt ttatccataa ttgtttcaaa gcatagtata 4980 acagaaaatg tagccggctg agcaactttt tcagttcatg atagcaatca ggccttgaat 5040 ctaactaaca ccttttatat acttcagata atacgcatga tactctttat agttacattc 5100 ctctccattt acggcctgat ccatctgtat gcatttgtaa aggcaaggtc cgccctgcgg 5160 tttggtgcag cgcgcggggc agttctcggt cttttcatgg ccttcatggt cttcagtccc 5220 ataatagtac gtacctctga gcggctcggc cttgagacct ttgccgtcta tttctcatat 5280 gcgggatata tatggatggg cctccttttc ctatttatct catggtctct tcttttcgat 5340 atctgccggt tcatcgtatg gttattcggc cgcgtatgca ggtataatct tacggccatg 5400 atgcctccgg caaagtcatc tttttatctg acgctcatcc ttacgcttat tgccgctgca 5460 tacggatcgt atgaggcggc agacattcgt acggagcgcg ttgttatcaa gaccgataag 5520 atgccgaaag agaacggcag gttacggata gtacagatat ccgatgtcca tatcggtctt 5580 atagtaagag aatggcggct tcagagaata gttgataagg tcaggaacgc cgggcctgat 5640 attcttgtat ctaccgggga cctggtggac ggacagatcg acgggcttga cggactcagc 5700 ggacttttga agggcataga cgcgccgtac ggaaagttcg cgataccggg taatcatgag 5760 tattacgccg ggttcggaca gtctgcagaa tttatcaaaa attccggctt caggcttctg 5820 gaaggggagg cggtaacgat cccgggattg ataaatattg cgggtgttga cgacccggct 5880 gcaaggcggt tcgggctgta taccgctatt ccggaggagg atcttctttt gtcccttgac 5940 ggtgagctgt ttactctgct tttaaagcat ctgccgctta ttaatgagga ctcgttgggg 6000 aggttcgacc ttcagctttc cggtcatacg cacagggggc agatattccc gttcaaattt 6060 attgtgcggt ttttcttccc gaggctttcc ggttattttg aacttgaaaa ggattcccgc 6120 ctctatgtca gccgcggcac cggcacctgg ggcccgccga ttcgctttct ctcgcccccc 6180 gaggtgacgg tgatcgatct ggtgggtgag aaatttcagt aacagttcag tatgccggcg 6240 atttcctgtc aatgtttctg ttaaaattag atcagtcaga aaattggtta tcagtgtata 6300 tttcaggaga aaagaaagaa tgaatcttga gatcagggaa atgacgatcg acgacctcgc 6360 gccggtattc catcttggag aagaactttt cacgtcggaa agtctctcga acttatacag 6420 gacatgggat gaattcgaag tgaccgcctt ttatcaatca acaccggaat attgcctcgt 6480 tgccgatgat gacgggaagc tggccggttt tctgctgggc acgacgatcg agaaagcagg 6540 aacagcctgg aactacggtc atctcgtatg gcttggtata cataccgaat atcagcgtca 6600 cgggatcggg aaccgtcttt ttgaagccta tcgacagttg atgataaagg aaggtatccg 6660 tatcatcttt gtagatacac aggcggataa tgaagcggca gtgaaatttt ttgaagacct 6720 gggatttgcg agggggacag agcatgtgta tatgtcgctt aaccttgaca ctgatattaa 6780 aacgtagtgg aacggcacga aaaggagagc cgttatttgg atcacattga tacagagcgc 6840 cttagatcat tgttcaaggg gttgactgat atatacagcc catcgggaaa agaggtcgaa 6900 gcacttgaat atgtcgaacg gttccttgat aaatatttcg ttccttaccg tcgtcttcct 6960 gttgatgata agcggtacaa cctgtttatc ggccatgatc ccgttgagac tgaggtgctg 7020 ttcctggggc atatcgatac ggttcccgcg ttcgatcttg accattatca gatgacgata 7080 gatggtacga cagccttcgg cctggggacg gctgatatga agagcggctg cgcggcgatg 7140 atagaggctt ttacttcaca ctatgaacgt cggggcggga tcgcaggggc agcgctttcg 7200 ctggtagtcg gagaagaaga aacaggggac ggagccgcag cgctggtcga cgaggtccac 7260 ccggcatggt ccgttgttgc agagcctact gatatggtcc cggcccttgc gcattacggg 7320 tatatcgaga tagagctcga tacctccgga gaacgctccc atgcatccat ggcaaagacc 7380 aatgacagcg cggtcaagac catgctcaat ctccttttga aaatgagtca gcaccttgat 7440 gccgcgcatc cggagctgat ctgcaatatc cgtgatgtgc acagctcgga aggagggttc 7500 gcggtcccgg acagctgcag tgcctctatt gatctgcacg taccgcccaa ggccaacctg 7560 gggaagcttg tctcggaaat agaggagctt aaccagagca tgaatggacg gaaagccgag 7620 ctttcatttg agacagtcca taacggttat gaccttccga tgcaggggct tctacctgaa 7680 attatcacga aggtctataa agagcagggt ctgcctttga agacagcgcc tttcagaagc 7740 cactcggacg ccagtatcct gtgggcttca gggatcaggc ccattattct cgggccggga 7800 cagctttcca aggctcacac ctaccatgaa tccgtcgaaa ttcctcaggt cgaacaggcg 7860 gcacatatct attaccggat ccttcatgaa cttgagaatg tgaaagagtg atctttctat 7920 gttgccagta gtgtcaggtc ttcaccggtt tgagaacgaa tcagtgcctc aatgccgcag 7980 gcttctttca acgtatctta tggcagtgta agcccgatat ccggaagaag ttcgcttaat 8040 acaaaatttc tgtatcgtct cgccgcgcga atgaatctgc atgcatctgt tttttcgata 8100 gcggctgtct ttccgtgctc cagctctgct ctccttggac gccagagccc ttcatcatat 8160 ttgatcttct tgtctgcatg tgtcgtcttg cagaacgtgc ctttttcaag atgctccagc 8220 catttgtcga ttctcttttc tttggttcct tctgttttga gaatatcatg taccgcaggt 8280 tttattttcc cccaatcttt tccgctccct gcgtcaccat gtcggggatc aagctgtgca 8340 aaccttttaa aatattcatg gagccgcttg ctgccctcca ggaagtcctt gagattatca 8400 cggctgactt tcttcttcct tgaaccatct cttctcaatt catatgtata tccccaggct 8460 aaatacggac gatccggata tgtgccgaca gcaccatgcc caaccggtat tgcttcagca 8520 aatccgctga taaatcgtgt tttgaattct tcgtattttg ccttcatgta tcggattata 8580 ccgctggaat gtcttggtga gatactgagc gttccttcat caaccttatt gttatcatgg 8640 gaaataccaa tgaagccata gtgagcaaat gtatcggcat acacgtgggc ggcaatgcct 8700 ataatatggg gccagaacga tgcattcgcg ctgtccagag tgtaatccag catgttcctt 8760 gccgggacgc tgtccttctc acagactaac cggtcaatga agttttttcc tttataaccg 8820 ggcaggaaat gaaatgggat ccagaccttc cattgatcac ccggcagagc gttctgataa 8880 tcaataggct tgtggcttgt catggtgggc actatcgacc tgttgtcaga gagaacaatg 8940 tgatcatcat caattgagtc atcaacgaat tgagaagcat acgctatttt tccggctatt 9000 gaatctttta ttcccgcggc tcttgccagg gcataaactc cataaaagtg catatcgatc 9060 tgcatgattc cctccttttt taccgttctt gatataaggt ctttgctctg agatcatcat 9120 gtgatcaggt tgttccagat atgtattgca tacaatacag gaacgtgatc cttgatcgca 9180 tcaggtacaa gaccttgaaa accatggccg agtgtgctca tggcattgtt gaacctccct 9240 ttaacctcat ccgtccaccg ctcccagggg ctgatatcgt catgaatgtt tccctcactg 9300 tcgatatatt tcaatttccc tgcatgagca tacaagccgg gggccggcgg cggaactttg 9360 cagacaatat cattattatt ttcaaaacga tatgcgggaa cgcggaagtc ctttacaaaa 9420 tcttcatccc ccactctggg agacccaaac gtatacaggc cctgaacatt ttcaaatcga 9480 tacgcagcga gtgtggctat tgcagcaccg agactatgtc cggttatcca gagagcccgg 9540 ccctcattct gcagtccttt gacatagcta tgcaattcct gccagacctc atcaagtgca 9600 tcgctgaacc ccttatgtac tttccccttt ttcccggaat ctaccagccg gaagtcggca 9660 tccgccttga catcttcaac gatatcacga aaatcgtctt tatcctttcg ctttctgctc 9720 tctgttcccc ggaaggcgac aacaaggacg tctttattgc ttactgcata gcactgggtg 9780 cttttatccg taaaatatct gacctcagga aatcctgcct tttgaaataa tacccgggca 9840 tactcctctt ccgcatatac aagtgtcgcc gcctcgatca gccaccatgc atttgctatg 9900 ctgaattctt ttgtattaaa atcgaatgga taattttcac gcccggtaaa atactcgtag 9960 tctttatagg gcggagctat gttttcccat gttatctcgt ataattcttt ctttatcatt 10020 ttttctcacc agtgcctttc aaggattatt tgcccggaca ggcttttcaa gcaatatgca 10080 tacgacccag caaacttttc catatccccc ctcacgttaa aactgtttat tctggataat 10140 gtttacttgt atagaagaat cttatcatgt aatatgcggc actgcaaaag aggggagtga 10200 ggaggagctt gacgagtgaa tagactgtat ctggacattt tcaggggctg accccttcaa 10260 ataaaaatac tgtcgaaaaa ttttacgatt tattgtaaaa tgtaaaatac agtctcagga 10320 atgaagtcta acgtttaact ggaagtatga tataaatatg agagagaact ctgacagacg 10380 cagcagaccc acccctgccg taagcaagta tacattcttc ggcggccgaa gaaagaccgt 10440 gagaagggga tccgatacgc gcagatacct gtttgttgac cgctacgaga catcgctgtt 10500 tatagctgta atggcgctgc tgttgctgag ttttctggat gcctggttta ccatgattct 10560 gatcgataag ggcaaggtaa tagaggtgaa cccgatcatg tctttttatc ttgaatacgg 10620 tgtgataccg tttacgataa tgaagttctt tatcacggct atctccctga ccatcttctg 10680 catgctgaag gacttctctg tcaggagcta ctgccttgtg aacaggtctt ataagaacgt 10740 ctatatcatg aggagaagcc tgccgtttat aataaacctc tatttcatgg tcgttgtcta 10800 cgaagtctat cttctggcga tctaagaaat ggggtcaggc ttgcaaattg acattcttga 10860 gtagcatggc cactctctcc actttcattg aaagctgaac atgtcccctc tggctcgcat 10920 tcaatgtcca aaccgctggt actcgttcaa gaccatcaaa tgattaggca atgattgaca 10980 agccaagcaa ttaaaatagt ccttaaatat tactcaaaca tcattgcctt tttaattctc 11040 ttcatatccc tttttccatt cagcatttca agaagtttga acgcaacgtc taatgctgta 11100 tcaggccctc tatttgtaat aattctgcct gtaacgacaa ttctttcttt gaccgggatt 11160 gcaccacaat cttgaagaat cttcagattg tcatggcgtt tacttgcgga atatgtcgtt 11220 gcttttttac cttttaagat ccccgtcctg gcaactgcaa gtgaaccaac gcacatcgtt 11280 gctataattc cgttgttccc gtatattttt ttgataaggt caagaacttt ctgatggtag 11340 acttccgtat atccggcatc attaaatccg ccaggaataa taagggcctg ataatgtttt 11400 ggatttattt cttttatcaa ttgatgcgcc ttgatgttca gatcatgctt agaacggatg 11460 tttctcttga atgctgttat cacaagatcg acggaattta cttcttttaa aacccttgtc 11520 cagccgagaa tgtctgtgaa agcagcaagc tcaatatctt caaatccttg cggtgcaaat 11580 agtaatactt ttttattcac ctatccctct gattcatatt tattttatct tctgcctaac 11640 gccgaattaa ccgatgcgag cctgcatgat aatagcatgc aacttagtgc ttgacaacaa 11700 taaaacaatt gaatgcgaat taacgaccgc gggctcgcat tcggttgaat atctggttat 11760 acatttatta tcaattggta aaaaattgat tacatcatgt tatcaataat ctgcagggat 11820 tatacatttg aggattacac atgcttataa atgattttga ctcaaaaaag aaatgtttat 11880 tacagattga aacattcaga tataaaaaaa gggggaacga atcgttcccc cctttcgtga 11940 atgtattgct taacaactct cgccttcagg caactgattt aaaagttgtg tgatcaggtt 12000 atttgtgtaa gcaataacga gatttgctgc ctcatcagag atcttattgc ctctctgggc 12060 atcgacctgg tttttaagcg catcgagctt attgatggca gcacagatgt tatctttgct 12120 ggctgaattt tcagcgcttt ccaccttggt tacgaggctg ttctcaagtt caggctctat 12180 aacgccttca gctacaagtg tatttaatgt gctggtcatg ccgcttaagg tgtcaataca 12240 accgtcagtg tttgcatcta aaccagttgc atccttcagg gggcattggt caattgcatc 12300 gcccactgta tcatcgtcag tgtcaggatt aagcggatta gttcctatta cggtctcctg 12360 ctcgtcggtc aggccgtcgt tatcatcatc aggatcaaca cagtcagcat ctccgtcact 12420 gtctgtgtct gtaaagcctt cgtctatgac cccgctgcaa ttatcatcaa taccgttaca 12480 tacctctacc atacccgggt tgatcagagc ggcactgtcg tcacagtctg aactgtcagc 12540 aaccgcaccg tcaggtatgc cgcaggtggc atcaacagga aggttaatgt ctccataccc 12600 atcaatgtcc gcgtctgtat aagcagttgc cgcaggcctg tcatcaggtg ttatagcatc 12660 acacacaaca cccaccccgg cgcagatagt actgccgatc gaataatctc cgcagttgtt 12720 aggcagactg ctgcagacat cgccggtgtt aaacccctca tcgacatcac catcgcagtc 12780 gttatctgct tcatcacaca cctctgtgtt gccgggatag ttgtctgcat cactgtcatc 12840 acagtcatcc ccaggtgcag tctggcatcc ggcaggttca cagcctgttc cagcacaggt 12900 tccatcagct gatgcgttcg catgcccatc actgtcatcg tcacaatagt aatctgcttc 12960 gctcgaatac ggccttgcaa gtgggagata gtccggtcca ccggaaaaga catatgcact 13020 gtctacaaag ccatcgccgt ccgcatcagg agttgtccag ttgctccagt agttgccgcc 13080 cgtcggtgcc ggcagattga agacattgtt gtcactactt ctcatttctg tctgatgaga 13140 gttgcctgtg aagttgttgt tatagacctg gtttccattt gctatgttga aaaataatcc 13200 ccaattgttg tacgacacgt tattctctgt caaagtgttg ttatcgctaa cgctgaacaa 13260 gactccatac atactgttag agatggtatt tcctgtcagg gtattgttgc tagatctata 13320 acaaaatatt ccgtaattgt tgcttaagat gctattgtct gtaatagtgt tattgctgcc 13380 gtagaacata cggaatcctg tatgaccgtt ggaagacaat gtattacctg tcacagtatt 13440 atcgttcgaa aggacatcga acatgaatcc ggcaataagg ttattcgtgg cggtgatttc 13500 cgtgagagtg tttgcgctgc tccaattaag gaagacccca taggtaaaat tcttaacaac 13560 taaattctta atggtcactc ccgacctgga aggtatgtag ataccgaatc cggagcctgt 13620 gccctgtatt gtgtagccag ctccgtcaag ggtcacatta taactctgaa tctgtatgga 13680 ttcgtaaacg tttgtgtgta gagtacaggt ctttgagggc caatcccagg tgcctatata 13740 agtacagtcc ccgcctgttg cattatcata tataaacttc gtgctcggat ttggctgtag 13800 ccagacgtag tgcggacccg agaggtcctg ggtccaggta taactcccgc tttcgtccgt 13860 tgttatggtt tctccgtggt catatccgtc ctcatacttg taatatgttg ttgactgagc 13920 aaggcctccg agggtcatct gagacgatgc cgccccttcg gacgcttcaa tattcattgt 13980 tatcatcgaa gccgcagact ccagcgtaag gcttattgac tctgtgctct ggagcgtcac 14040 gttgagatat tcactgtccg tgacctcaaa ataagtgcca gtgccctcaa ctctgcccat 14 100 tatcgatgtt acctcactaa aaggttcagg gtcttcggcc acagctggtg ccatgcctga 14160 aagcaggagc actgctaaaa atactacagc aaaaaaaagt ttcgttttca tgttatcccc 14220 cttaaagtcc cataaattta gaaattaaca tattaaactg atagataatt cagaatttac 14280 cccctttcat acaaatgaat attcgcgtat cacgaatcag aagcacatta agttatgtag 14340 agataattca ggtaagtgag aacagacgag tttttgatat ccctaaccta taaaatgcaa 14400 gcaaataata taccgattat tgaaatgctt atattatgag gagttagtag aataaaaaat 14460 caataaaata tgtcatcttg gcaaaattac caagtcactt tggcaatata actatctgaa 14520 aatattagtt atttagaacg attctaaatt aaaatatagt tgaaaatcac tctcactgaa 14580 acataaggat taacgactga aatcttaagt gctttgtaaa gtgaagcttt attgataact 14640 tcaacctatc aataagatat agactgattt aaccccatca ctaatagagg tttgttgtta 14700 accattagac agaagctttg aatccttcgt gatgaataat gtactatatt tcatatgtgt 14760 atataagagt gtgatattca tcatcccttc ccaaaatagc ttctaaggcg gtttggaatg 14820 aaaaaatatg gatttgcgtt agttgttgct gctgtaatta ttgtcattat catgttccca 14880 tcagataaga aacacatacg aaaaacgatc aggacctctg aacaagcctt tcagataaga 14940 aacacatacg aaaaacgatc aggacctctg aacaagcctt tattaaagaa gaccttaaat 15000 ctgtaatgga tttcgtttca ttgaattata ccgatgatta cggaggcagt tatttgacgt 15060 ttaaaaaaag ggctgaaaga ctctttagca cttatgatga ttttgaggtc tcttcggata 15120 tagttgccat tacagtcagt ggagacgaag cttttgcgga tctgagagta agccttattg 15180 catcagccgg aaatgaaagg ggctacctgt ttggggatgc cggaagccac cgggagataa 15240 gagtatattt gggaaaagaa aagttcggtt ggatgattgt cagacttgat gactatagtg 15300 aaaaatctat aaaatctagc 15320 <210> 4 <211> 15364 <212> DNA <213> DNA sequence 2 of pFosLipG <400> 4 acacaaacca cgaactcgat atggcgtgac atcaaggtct tcgagaatca cgcatacatc 60 gtcgctgacg gcgcaggcgt ccatggcatg caggtcttcg acctcactcg actgcgcggc 120 atcgcgtccg cccaaacgtt cggtgccgac gtcgtctacg gcgacttcgg aggcgcccac 180 aacctcgcca tcaacgaggc gacgggtttt gcgtatgcgg tgggcacgaa cacctgtcgc 240 ggcggcctcc acatgatcga tatttcggtc ccgaacaatc ccctgtttgc cggctgccac 300 gattccagcg acacgcacga cacgcaatgc gtttcctatc aggggcccga tgccgaccat 360 gccggccggg agatatgcgc gagctccaac gaagaccacg tcgaaattgc cgatgtcaca 420 gccaagaact cggcgcaaac gctggcgacc gcggtctacc cccagctcgg tttcgtccac 480 cagggctggc tgaccgaaga ccatgtccac ctgctgatcg gcgacgaact ggatgaaaga 540 aatttcaact cgccgacacg gactcacgtc ttcgacgtgt ccgatctcga caatcccgtg 600 tacctctacg ctttcgaggc aacgacgttt tcgatcgatc acaacctgta cgtgctcggt 660 aaccgtatct tcgaggcaaa ttacacctcg gggctccgcg tgctcgaatt cggcgacctt 720 gccagccagg agatcagtga atcggcctac ttcgatactt ttccggtcgg cgatgagagg 780 gaattcgacg gcgcctggag cgtctatccc tacctcccgt ccggcaacat catcgtcagt 840 gatgtgtcga acggcctgtt cgtactgacg gtccaatgac gcgcgggtcg ccgcccgcgg 900 gcgaccccac cggggaaacg ctcaggcagg cggcgcctgc tgctgccgtc gcgtagcaat 960 ccgctcctga agctcgtcaa acagttccgc ccgcagcccc agaatcttga aaatttccac 1020 gatctgaaaa aatggcgcgg ccaggttcgc ctggaccgga tgcttgacga gcacgggcat 1080 cgacttttcc acggcatggc cgatgaactg ggcggcgtag ccaccaaaga atgccgccgc 1140 cgcgatgcct cccgagacgg caatcggctg ctcgccaatg acaccggcta gctgggccac 1200 gagcatggcc agcaccagga acgcaatggc gaaccagcga tccagggtca ggtagaagag 1260 gaagaaaccg atcatcgcgg catgtgccat attgacgcgg atgccggcga tgtccgcgcc 1320 cagccaggtc agcgggatca gtactcccag catgatcgtc ggtatgccga tcatgtggac 1380 agatacgttg aacgggtgct gatgcgaggc agcataccct gtcagcatat ccatcaattt 1440 tccgtcgttc ttggccatcg agccattata atcacaagtt cagggtgtaa cgggaagcaa 1500 cgagatgagt ctgctgattg tgggtattgt cgtttggtgc atcgtgcatt tgtttccctc 1560 catcgcgccg acccggcggc aggcactgca cgcccggctc ggcaacggct atcgaggcct 1620 gtttgcgctc ctgatactcg cgtcgctcgt gctcatcgtt atcggctggc gctccgccgc 1680 gccgtcggcg gtctacgtcc cgccgctctt gggcagcccc gccgtttcgg tgctgatgct 1740 catcgccttt gtcttgttcg tggcggcccg cgccaagacc aatatcaagc gtttcctgcg 1800 ccacccgcaa ctgacgtcga tcattgtctg gtctgccgcc cacctgcttg ccaatggtga 1860 cacccgctcg ctggcactgt tcggcagtct cggcgtctgg gccatcctgg agatcctgtt 1920 gattaacaaa cgggagggcg cccgggacaa gccagcgccg gttcccttaa cgggcgatgt 1980 gatcaccgtt atcgtcggcg ccgttgcctt cggcgttatc gtcttcagtc acgaatacct 2040 gtttggcgtc tcggcgctga tgtgacaggc ggagagatcg atgagtctgc ccgtcctgct 2100 gttacttgct ttactggtac ttgcggtctg gctgcttatc cgtctcaggc ggggcagcgc 2160 cagcaaaacc gagtctgcca acccggtttc gaccagtgaa acgtccaaat ttcatgcggt 2220 ttcgatcaag gttccaaaga ccgcgtgcgc ggccgccaag gccttgacgg gcgagcggtt 2280 cctgtcgacg gaagcaccga cgttaccctt gcccggctgt gatgcgcccg actgcaactg 2340 ctgcttcgtg catcacaagg atcgccgcag cggcaaagac aggcgtagcc cgttcagccc 2400 gggagggttc ggcggcggca cggggaaatt cgagcaggaa cagcgcagga gaggcgaccg 2460 ccggaagacg gacgacgagg acttcttcta aaagccctcg ccgcctgttg cattgaattc 2520 agccgggact agtaaccctc tgcggcgacc gtctgttcaa aatactgcag ttcctgttcg 2580 gcacgcgcgc gttccgcgac gaggaactcg atttcggatt cgagttcgcc cgtgcgttcc 2640 gacagatctt tcatctccgc gagaagctct acgcgctgct cggttgtcgt ctcgtcactg 2700 atcagttcga caccgatagc gactatcctc ttctccgtgg acttgagttc cccctgcttg 2760 gcatagatgg ccgaattcgc actgctgaca gcggcacgta gcgagtacag ctgatagccg 2820 acgttgtagg aggcaacgaa ctccggctcg agatcggcag aacagacgcc gttataacga 2880 ccaccgttga cgccgtatgt gaagccgtgc ccgggctggc agaattccac gaggccgtcc 2940 tcgcggccac gcttgtagcc ctcgaagtct gccgcaacgc cgtgcttggc acaggctttg 3000 cgatgttggc tgaatcgatc ggcggtgtaa ccccgcgcgc cgtcctcgta accgatcgcc 3060 gaccagtcac tgacgaggca ctcgtcggca ctcatcgacg cgcagccact cagacccaga 3120 atcattgcgg ctgcaagaat atttgccagt ctgctattca tctcgaaccc ctcgaacttg 3180 tctcgtgttg attctgaatc acttttaccg ctgcgttcct gaatccaggc tgaaaagtgc 3240 ggcatgtctc gctttccgca ggcacccggt gtgacctgtg tcacacctcc gacgggtagc 3300 gccggataac ctgaaccacg acagttccgg ccgggtcaag aagcatggac attcacggat 3360 tcagcggcgc cgctaccgtt gccctggcca gcgcgacaat cttcgtgctg gttgccaggt 3420 cgtggctcat cctgtcgcgc tcggtacgta ctcagcacac tttcgccgac ggcatcatgc 3480 gcgaggcggc acagcgtttc cgcgacgaat tcgagcggct gagcgccaag gagtcgatct 3540 atcttggcgc aggcctcgtc tttctcgtgt tatttgccgc ggcctacgaa ctcgaggcgg 3600 agcggcttta tcttggctac cccctgtggc agctatacat cctgttcgtt gccctggccg 3660 cttgcctgct gctggccctt cagcaactcg tacaaacggt cgcagagcgc aacaggatca 3720 agctgctacg tgatgcgaac attgccgtcg ggcaccaggt acagcgaatt gccgcgggcc 3780 tcggggccgc ttaccacgat gtcgagacga gcgcaggaac catcgaccac ctgatcgtcg 3840 gcagtaatgg cgcctatgcg atccacgttt ttgcccggcg acccgtcaac aacggcagcg 3900 ttatgctcga cggtacggag ctggtcttcg atcctgccga aaagacggag tcgatcgtcg 3960 ccacggcggc cgcggtggcg gcgctcgagc gcgagttccg gcggctcctg gatcatcgcg 4020 tgcgcgtacg ctctgtcatc gccgtccccg gctgggaaat caagggtcaa tccggcgacg 4080 agcatctcct ggcgaacgag aagaacctcc ccatgctgcg cggctggaaa gacaacaccg 4140 accacctgat ggacgaggac gtcgaggcat tgcacgcctt actgatgtcg cgctgcagac 4200 tcgacggcaa acgcgacagc cgcgccatcg agaagtcaat ctaggggcac gagtcgggct 4260 tcgagttcgc ctttatcgat cacctcgtgg aattcgtccg ccagcgcggc gcactccgtc 4320 acgacttgcc gccagatgcg tatccgctcc gcaggggcat agcgctggaa gtcgaaccgg 4380 tccggaatct tccctttcgg cagccgcgcg acgaattcgt cggacgggca caccagtatt 4440 gtccggctga cgtgctccgg gcgcggcttc cgccaggtaa ggcgcttgtc gaaccagccc 4500 gggaccatat ggccgtagaa atgcggaaac agggccaggc gatccggctc gctgtgcggg 4560 acatcgacgt gatagtcgat gatgcccccg tctcggtaca cgcccggcag cgctcgcggg 4620 acattacgga caccgctgag aaccatcggg atcgacccgg tggcaacgat cgcgtcttcg 4680 acgttgtccg ggccgacgcg aatgcgttgc agtgggaagc cgccaacgtc gaagaacggg 4740 gggagatcgc gtgcgtcgaa aaacagggct cgctcgaaga atgcggccag cagacggcga 4800 tcaatcacat tgagcgaggc cgccgccagc agggcggtgc cgagaaccgg ccgctggtcg 4860 aatgcagcga ggttgcgggc acgtacggcc aggatgtgcg tgcgcagcac cggatgatcg 4920 agaacctccc tggcgccggc gtcacccaac aagacgcgca gaatctcacg gctcttggcc 4980 gttatctcgt gaatatcggg cctctcgctg tagctttgct ctatgtacgc gtcttcgaag 5040 cgttcgaccg cggcaaacgg atcttgctga gcgtagcagg caaagcgcca ggcgccgatc 5100 gacgtgccga tgaggtgtac cggaccggat aatcgaggta agaggctgtc gaatatggcg 5160 cgatccaact ggctgagtac cagccatttc gcgccgcccg aagcgccgac gagtgtgcct 5220 attctttccg gcgcaaaacc gtgccgctgc acgctctgca gcgcctccgg accagccttg 5280 aagacgagct tcggattcgc catgccggcc cgcgcctcgc gagctagccg aacggatgct 5340 gcttgattat cgtctgctcg cggtccgccc cggtggaaat gatatcgatc ggcactccgg 5400 cgagctgctc gatgcgcgcc aggtagtcgc gcgcaccggc gggcaaggca tcaaactcgg 5460 tgatgccgac cgtcgaggac cgccaccccg gccattcctc atagatcggc tcacactccg 5520 caaaccggtc cacgaccacc ggtacccctg cgatcggctc cccgtcgatc tggtagccca 5580 cgcaaatctg gatggtctcg agctcatcga gcacatcgag cttcgtgacg cagagtcccg 5640 acacgctgct gttgatgatg gagcgccgaa gagcgacggc atcgaaccag ccgcagcgcc 5700 ttggacggcc ggtcgttgca ccgaactccg caccgacccg ggcgagatgt tgcccctggt 5760 catcgaaaag ctcggtcggg aacggcccgg aaccaaccct ggtagtgtag gccttgacga 5820 tgccgagaat gtagtcgaga ttgcgcggcc cgataccggt accggtactc gccgccgcgg 5880 cgaccgtgtt tgacgaggtc acgaaaggat acgtgccgtg gtcgatgtcg agaaacgttc 5940 cctgggctcc ctcgaagaga atactcttgc cggcatcggc cagctcctgc agcagttgcg 6000 tcacatcggc cgtgaccggt gcaatgacct ccgcggcttg cagcgattca tccagcgttt 6060 ttgcaaaatc gaccgggtca gcacggaagt agttcttcag caggaagttg tggtagtcga 6120 ggacttcgcc gagcttggcc gcaaagttct cacgcacaaa cagatcggac accttgagcg 6180 cccggcgggc cactttatct tcatacgccg ggccgatgcc gcggccggtc gtgccaattg 6240 cggcggcgcc cttggctttc tcgcgggcca gatcgagggc ggcatgcgat ggcaaaatca 6300 gcgggcaatt ggggctgatc ttcaggcgct cgaacacggg gacgccgctc gcgatcagcg 6360 cgtcggcctc ttccaccagc gcctccagcg acaacacgac gccattgccg atcaggcagc 6420 tcacgccatc gcgcaaaatc cccgagggga taaggtgcag cacggtcttc ttcccgtcga 6480 tgacaagcgt gtgcccggca ttatggccgc cctgaaaccg aacgaccgcg gcggcgcgat 6540 ccgtcaacag gtcgacgatc ttacctttgc cctcgtctcc ccactgagta ccgacaacaa 6600 cgacattctt acccatcagt ttcccgttcc atcagccacg gacaataaac agcaacacga 6660 ttcccgcgat catcgctcca agcccgaacg tgcgcagctg attgtcgctg agccgcacga 6720 tctgcgccac catttgccgg tagcggcccg ggccgacgaa gggcaggatg ccctcgatca 6780 cgaggacgag tgcaagcgcc gtcaggattt cgctccagta caaggtagct ccggattacc 6840 ggctgccttc cgactcgttg aggtaccgga agaagtcgct gtcgggatcg agaaccagca 6900 ggtctccctc gacaccgatg gacttgcggt acgcatcgat gcttcgataa aacgagtaaa 6960 actcggggtc cctgttgtag gcacttgcgt agatttccgc ggcgcgggca tccccctcac 7020 cgcgtatctt ctgactgtcg cgatacgcgt tcgccaggat gatcgtcccc tcccggtccg 7080 cctccgcgcg catctgctct gcgacctgct cgcccgtcgc gcggcgttcc gttgcgattc 7140 gcgcgcgctc tgcggccatc tgctggtaaa ccgagttacg cacgtcctcc atgaactcga 7200 cctgcttcac gcggaagtcg atcagttcga caccgaggtc catagctgca tctgccgcgg 7260 tctcgcgcat atcccgcatc aattcctgac gtcccaccga gatcgcgtcg ttcaccgtgc 7320 gtttaccgaa ttccgtgacc acggcgttct tgatgatctc cgaaagacgc ccgttggcga 7380 cttccaatat gccaccgctc gacgtgtaga aacgcacgac gtcgataatg cggaacttca 7440 cgaagaagtc gacttccagg gcctcgtttt ccgccgtgaa cacacgctcc gggcggtcag 7500 aaatcgtcag gatgcgtttc ggaaacttgc ggacattctg gaccaccggg atcttccagt 7560 gcagaccagg ttcatattcc gactcgacga cttcgccgag ttgtaacttg atcgcgagtt 7620 cgcgctcatt aaccgtgaat gcagaaagcc cgacaccgac gagcaccaaa cccaatatga 7680 ctaccaaacc gaatcgcccg ccgttcattg cctgaccctc ctctcgcgtg gatcgactgc 7740 cggatccaac ctcgggttcg gctgcgactg cgacggtgtc tgcgagtctg cagaccgggt 7800 gctgtctgag ctacccgacg gtcgagttgc aagattcaac attttgtcga gcggcaggta 7860 cagcaaattg ccgctcccgt ccgagtcgat gaagaccttg ctggagcggc tgtaaatatc 7920 ttcgatggcg tcgatgtaca gccgttccct ggtcacacgc ggcgccttct ggtactcctc 7980 gagtagcagt tcaaagcggg cggcttcacc ttcggcatcc gcgatgaccc ggtcccggta 8040 ggccttggca tcttccagca cgcgcgctgc ctcgcctcgg gccctcggaa ccacgtctct 8100 cgcataccgg tcggcctcga gctgaaagcg ctcactatcg ttgcgcgctt tctgcgcgtc 8160 gtcgacagcg gcttgcaccg agtccgggta gttgacgttt tcgagtgaaa tcgacgtgac 8220 cgtcagccct gccccgtagg aatccagcgt gctctgcagc gcctcctgcg tgcgcgaggc 8280 gatctcgtct cgcctggccg cgatcaggac ctccagttca ctggtgccaa cgacctcgcg 8340 cagcgcgctt tccgtgacgt cctgcagggt ctgctccgga tccgcaacgt tgaaactgta 8400 ggccaccgga tcggtgcggc gatactgcac gaccatgtcg atgttcacgt actgctcatc 8460 ggccgtcagc atctcggtcc ggtaggcgta attggaaacc tggtttgcgt tcaccagatc 8520 gaccgtttcg atcggaaacg gcagatgcca gtgcaagccc ggcatggtcg actcggtgta 8580 ggccccgaat cgctggacaa ctccacgttc ggcttcatcg acccgataaa aacccgtgag 8640 cccccatgcg acaatcagga ggatgacgag aatataaccg cctccgccgc cgcccgagcg 8700 cgccccgcct ccaccaccga gaatgccgct cagtcgcctc tgccagtttt gtacgatttg 8760 atctaggtcc gtcggagcgc cgtcatcgcg cttccacgga tcctttccgt ttcccgaatc 8820 attccaagcc atgttcgtct gctttatcgt cctgagttta tgccgtcgcc ggttttctgg 8880 tcggctccgg ctccaaaatt tcggccgaga gattctcgcg cttgagaaag cgcatcaaat 8940 cccgctcagc catttccagt tcaatcgtcc acccgccatc ttcgcaggtc tcttcactga 9000 ggacagcacc ggcttcgaaa agtttagccc gctgccgcgc ctgcgtgggt gcaagatgta 9060 tggtgccgtg caccgtctgt ggtcgcagcc tttcgctgat cgcttccagc aggaacggaa 9120 cgccttcgcc ggttttcgcg gatagccaca ctgcacggcc ctcaccgtgg cgattattcg 9180 ccactctggg tctgcggtcc agcatgtcga tcttgttata gacgcggatc tgcggcacgc 9240 ggtctgcatc gagttccttg agtacggcat tcacctggcg aactcgctgc caacgactac 9300 cgtccgatgc gtcgatgaga tgcaggataa ggtcggcctc tcgagcctcc tgaagcgtgg 9360 accgaaacgc cgcgatcaac tcgtgaggaa ggtccctgac gaatcccacc gtgtcggcta 9420 gcacgatctc gcgcccatcc ggcagctcca gccgccgcac ggtcggatcc agcgtggcga 9480 acaacttgtc ctcgacgtaa acatcggcct gcgtcagcgc gttgaacaac gtcgacttgc 9540 ccgcgttggt gtagccgacc agggcgaccg tggggacttc ggcccgcact ctgttctgcc 9600 tgttcatcgt ccggcgagca tcgacatgct cgaggcgctc gtgcagttgc cggatccgct 9660 tgccaataag cctgcgatcc gtctccagct gcgtctcgcc agggccgcgc agaccgatgc 9720 cgcccttttg tcgttcgagg tgagtccagc cgcgcaccaa tcgcgtggac aggtgctcga 9780 gctgcgccaa ttccacctgc agcttgccct cgaagctgcg cgcccgctgc gcaaatatgt 9840 cgagaatgag cccggcccgg tccagcacac ggcacttcag ctcccgctcg agattgcgct 9900 cctggctagg agtcagctcg gcagaggaga tcacaagttc cgcgccgtgc tctctgacgc 9960 tctgcgccaa ctcgtccagc ttgcccttgc caatgaagta acgcggatcc ggtcggcgcc 10020 ggccgcttac gatctgatcg acaataatgc caccggccga gcgcgccagc tcggcaaatt 10080 cctcacgttc gcttgcgtcg ggcgcaccca ggaggctggc atggacgaga attgcacgtt 10140 caccgctttg cggtcgttca aacaataacg acctcccgcg gctgtctggg aatcaaacta 10200 gccgtctccc gcctcgtcgt ccggcaacgg caggcgaacg tttctcgacg gcaccaccgt 10260 cgatatcgca tgcttgtaaa ccatctggtt gacgctgttc ttgagaagca cgacgaattg 10320 atcaaacgaa tcgacctgtc cttgcaattt aatcccgttg acgaggtaga tcgaaaccgg 10380 gaccttttct ttgcgcaaaa tgttaagaaa gggatcttga agcgtctgcc ctttagccat 10440 gtcggggtct ccttagttct tgttgttctt agaaccgctt tccggtggcg ccggttgccc 10500 aaagcgagtt cagcctaagc gttgaaaaat tctatcagag acttcgatat gccgtcgatt 10560 actccggatt caagtgggtc gaagacctgg acacctgact cgctgcgcag ccaggtgatc 10620 tggcgctttg cgagctgccg ggtcgcgaac aaggccttgt ccctcgcctc ctcgacagag 10680 acgcggccgt cgaggtactc ccagacctgg cgatagccga ccgcacgcat tgacgggtgt 10740 tcagaagtca ggcccggcct atcatataaa gctttcactt cctccaataa cccattgtta 10800 aacatagttt ttaagcgagt ctcgattcgc tcgtgcagta aacttcgcgt cgccggaagc 10860 agcgccagtt tgcagaattc cacatcaatc atgcgtcccc tgccgctccg cttctgccat 10920 tcgctcagcg tcgtaccggt cgacaggaac acctcgaggg cccgctgaat gcgctgtcgg 10980 tcgccgggtt tgatgcgtgc tgcggcttcg ggatcgacat ccaggagttg ctgatgcagg 11040 gcctcccagc cgattttgtc cgcattcgca tcgatctttg cgcgtatttc ggggtccgcc 11100 gcgggcagtt ccgcgatccc ttcggtcagg gcacggaagt acagcatcgt cccgccgacc 11160 agtaacggga tgcggcctgc cgccaatatc gattcgattt ccgcgatagc atcccgcacg 11220 aagtcgcccg cggagtagct ctcttcgggc tccctgatat tgaccaggcg atgtggcgcg 11280 cggcgcaggg tctcggcatc cggcttcgcc gtgcctatct ccatgccccg atagacgagc 11340 gccgaatcga cgctgatgat gtcgaacgga aaacgcttgc acagccgaat ggcgacatcg 11400 gtctttccgg atgccgtcgg ccccatcagg cagactgctt gccgcacgtc gccccgccct 11460 cagcgaccgc gcaagaagag gcggtcgagt tcggccatcg aaatggctgt ccacgtggga 11520 cgcccgtgat tgcactggtc tgcccgttcc gtggtctcca tttcgcgcaa cagcgcgttc 11580 atttcctcga gtgtcagctg ccgattggct cgaaccgagt gatgacaagc catcgtcgcg 11640 aggaactcat ggcactcgtc ctcgacgcgg ctgctctgac cggcctgggc gatatccgcc 11700 agcacgtcgc gcagtaatgc ctccgcgtcc gcgcccctga gaagtgccgg cacctcacgg 11760 acgaccaggc tcgtcggccc cgcccggtcg acgacgagac caacgagggc cagtaactcc 11820 gatgcctctt cggcatgccg ggcttcactc tccgcaacgg ccacggtgac cggaacgagc 11880 agcggctgcc gaaccagggc cttgtcgtca aaccccttct tgagcttttc ataggtaatg 11940 cgctcgtgcg ccgcgtgcat gtccacgacg accaggccct cccggttctc ggccaggatg 12000 tagacgccgc caagttgcgc gatggcatac cccatcggcg gtaactcccc ggcatcgcgg 12060 tccgacgccg acaccggtgg cgccccgccg gccagcgccg aataagtcgc gagcgattcg 12120 cgtacggcgg ccgggcgcgg cgcggccggc agccgcaggc cgtcttgtcg aatcggcgag 12180 ccactggcga ccggcgccgg cgcgacgcca tggccgctcg gccgggtgtc actgagggca 12240 acttcgaccg cctgtgccac aacgccgtga attcgccgtc cgtcgcgaaa gcgtatctcg 12300 tgcttggccg ggtgggcgtt ggcatcgatc gtcgccggat ccaccgtcag accgagaacg 12360 taagccggga accggccatg aaacaggacg tcgcgatacg catggcgggc cgcgtgactg 12420 agggtcttgt cactgattga acggccattg acgaaccaga attgcaggtc cggctggcta 12480 cgattgaacg tgggcaagcc aatccagccc gttatcgcga tgccttccgt ttcatgctgg 12540 agatacaccg cctgttcggc aaatgcctcc ccgcaaatcc tggccaggcg ttgctgctgc 12600 tgctctcggt tggttgccgc ccgcaagtcg agcaccgtgc ggcgattatg ggtcaacacg 12660 aaggcaacgt ccggcctcgc cagcgcgagg cgccgcaccc atttatcaat gtgtccgaat 12720 tcggtccgtt cggtctttag aaagcggcgc ctggccggcg tgttgtagaa caggtcgtgt 12780 acctcgaccg tcgtgccgcc agggtgcgca gcgggccgcg cctcgccgat ctcgccgttg 12840 tcagcttcga cctgccaggc gttgtcgcca ccggttgacc gggaagtcag gctcagccgc 12900 gccaccgacg cgatgctggg caatgcctcg ccacgaaaac cgagcgacac gacagcctcc 12960 aggtcttcca ggctcgaaat cttgctcgtc gcgtgccgcg cgagggcaag agacaattca 13020 ccctggggga taccggcgcc atcgtcgcgc acccgaatga gtttgctgcc gccggccagg 13080 atatcgacgt gcactgcttg cgctccggcg tcaaggctgt tctcgagcaa ctccttgacc 13140 acggacgccg gccgctcgac gacctcgccc gccgcgatct ggttgatcag gtgattcggt 13200 agctgctgga ttggcatcgt ggagacaaga attcgggaca atccaccatt ttttcaaaac 13260 ggcggacaaa tgtcttctcg aaggagcggt tatcaggtcc cggagaatac cggaatggac 13320 agcgtttgcc cgacccgaat cctgtcactg ctgagccgat tggccgcccg aatggcagcc 13380 gtgctgacct ggtagcgctc ggcaatctcc gacaacgtgt cgccgcgtgc gatgacatgc 13440 cgaacctgcc gatcgggggt ccgccgcaca tccatcgcga tttgcgtgtc aggtggcgga 13500 ttcgtgtaaa agtaattgcg aattcccgac aggatggcgc tggccagttt cgcctggtga 13560 gcggcgtcgc gcagcttctt ttcttctgtc ggattggaga tgtacgccgt ctcgaccagg 13620 atcgacggca tatccggcga tttcaaaacg agcagcccgg cctgttgcac cgtcttgcgc 13680 cttaccttga cgatcctgga cagttcgccg atgactttcg aaccaacgtc gaggctcgca 13740 ctgagagacg cgttctgtga caggtcgagc aagacttccg ccaggaccgg atccttgtcg 13800 tccagcgaca ctccacccac acgtaccgcg gcgttttccc gttcagcgag cagccgcgct 13860 tcctcgtcac tggcgccttt gagcgacagc gcataaacag ttgccccgtt cgcgcgacgg 13920 tcttcgacgg catcggcatg aatcgagacg aacaggtcgg ccttgtgttt tcgcgcaatg 13980 gccgtgcgct ggcgatggtc gacgtaatag tcactgtccc gcaccagcac ggcgcgcatg 14040 cccttttcgg cattgatgcg cgaagccaac tgacgagaga tcgccagggc gacgtctttt 14100 tcccgcgtcc tggccttgcc gatggcaccg ggatcgtgac cgccgtgacc cggatcgacg 14160 gctattacga tatcgcggcc cggcgtatag gactctgagg cacgtttaac cgtgtgcaga 14220 ctgcccgtct gctgcagatc gatgacgagc cggtcgccat attcgctatt cggccccgcc 14280 gtgaaactgc gcgagcgaac gtcctggctc aggtccagaa cgacgcgcaa ctggccgttt 14340 gcatttccgg tcctgatccg cttcaccgcg cccgcaccgg caggcaggtt ggtgaggccc 14400 gcgccgagcc ggccgtcttt gagatcaacg acgagccggt caggtccgcg aagggtaaaa 14460 atgttgtggc tggccgggcg actgagatcg aggacgacgc gcgtcttgcc gttttcagac 14520 cagattcgaa tgttctcaac ggtcgttgcg gcctgaaccg cgctgacgca gagggtcagg 14580 agcgcagtca gaatcaggcg tcggctgtgc attgcgggcg atcgctctca cgttcgggga 14640 ttatgtcggg tgagtatacc gtcacgatcg ggaattccaa gatttttttc ttgcaaatta 14700 gcaggataac caacttactt aacacgggaa ctaggtcttg tgtttaccat cccatgaagc 14760 cacaagagct tggccacgct cgctcagtcc gaggatttcg acgtctctgc cggctccgga 14820 gtatcggagg tgtatgcgca ggtcggccgc ggcggccagg ccgggcgccc gatccggcca 14880 ctcgacgaga cgcagaccgt cttcgagctc cgcccagccc agatagcgaa gctcttcctc 14940 gtcagaaacc ctatataaat caacatgata aactataccg ccgtcgaggt cgtagggctc 15000 gacgagcgtg tatgtcggac tgggaaccgc gccacggtgg cccatcgcct ggatcagtgc 15060 ccgggcgaac gtcgatttgc cggcgccgag gtctccctcg agcagcagcg tccatccgcc 15120 gacgtcgtca gggagctgaa ccagcagttc gctggcgagc gaccgcgtcg cctccgggtc 15180 gtctagatgc cgtttcatgg attgacgagt ccccggatct cgcccagcag gtcggatgca 15240 aggagaccgc gctcgccccg gcgtgccgcg aggtcgcccg ccctggcgtg aagttccacg 15300 ccggcggcgg cggcctcttc caccgagagc ccctgcgcaa gcagcgccgc gatgatcccg 15360 gtca 15364 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> N-terminal primer of lipG gene for PCR <400> 5 cccatatgat aaagaaagaa ttatacgag 29 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> C-terminal primer of lipG gene for PCR <400> 6 cccctcgagt gtgatcaggt tgttccagat 30

Claims (10)

Lipase protein having the amino acid sequence of SEQ ID NO: 2. A nucleic acid molecule encoding the protein of claim 1. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule has a nucleotide sequence of SEQ ID NO: 1. A recombinant expression vector comprising the nucleic acid molecule of claim 2. The pET22b (+)-LipG vector of claim 4, as shown in FIG. 4. A transformant transformed with the recombinant expression vector of claim 4. The transformant according to claim 6, wherein the transformant is Escherichia coli. The transformant according to claim 7, which is accession number KCTC 10985BP. A method for preparing a lipase protein according to claim 1 comprising culturing the transformant of claim 7 or 8 and recovering the lipase protein therefrom.  A method of non-specifically breaking down the ester bond of triglycerides using the lipase protein of claim 1.

Images