KR20090109364A - Novel gene encoding esterase derived from tidal flat metagenome and preparation method thereof - Google Patents

Abstract

PURPOSE: A method for producing mudflat metagenome-derived novel esterase is provided to massively produce the novel esterase and apply to various industries. CONSTITUTION: A mudflat metagenome-derived novel esterase has 10-70°C of active temperature. A polypeptide, polyepetide variant or its active fragment has esterase activity. The polypeptide is encoded by a polynucleotide of the sequence number 1. The polypeptide has amino acid of the sequence number 2.

Description

New esterase derived from tidal flat metagenome and its manufacturing method {NOVEL GENE ENCODING ESTERASE DERIVED FROM TIDAL FLAT METAGENOME AND PREPARATION METHOD THEREOF}

The present invention relates to a novel esterase derived from a mudflat metagenome and a method for preparing the same.

Esterase is an enzyme that hydrolyzes the ester bonds of triglycerides, and mainly hydrolyzes the ester bonds during catabolism, at pH 5-8 and 30-37 ° C. It has the best activity, and its decomposition products include acids (including inorganic acids), alcohols (alcohols) or phenols (-phenols) (-OH compounds) and thiols (thiol). Esterase may be useful for optical synthesis of bioactive substances such as tetrahydroisoquinoline, azabicyclononane, and indolemycine in the decomposition or synthesis reaction of ester bonds. In particular, it is a major enzyme widely used by its unique properties such as high substrate specificity and optical activity specificity.

Efforts to discover new enzymes, including the esterases were 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 the microorganisms in nature are not isolated and identified through traditional culture techniques performed in laboratories (Amann et al. al . , Microbiol . Rev. 59: 143-169, 1995; Hugenholtz and Pace, Trends Biotechnol . 14: 190-197, 1996; Ward et al . , Nature 345: 63-65, 1990). As a result, a new study is underway to create 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 to secure useful products. have.

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 cannot be cultured, but it is difficult to obtain information on the microorganisms from which the obtained genes are derived, but there is an advantage of simultaneously obtaining the products and genes of the microorganisms. The University of Wisconsin, USA, for the first time, has successfully isolated large-scale metagenomes and cloned them into bacterial artificial chromosome (BAC) vectors to build a metagenome library from which it has isolated a wide 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.

Accordingly, the present inventors isolated a new esterase gene from a metagenomic library of tidal flats in which various microorganisms exist, to produce a vector including the gene, and transform the vector into E. coli, and then, a protein produced from the transformant. The present invention was completed by confirming this excellent esterase activity and effectively expressing in water-soluble form.

It is an object of the present invention to provide novel esterases and variants thereof or active fragments thereof and polynucleotides encoding them which exhibit good activity.

Another object of the present invention is to provide a recombinant expression vector comprising the polynucleotide.

Another object of the present invention is to provide a transformant transformed with the recombinant expression vector.

Another object of the present invention is to provide a method for preparing a recombinant esterase.

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, the present invention provides a polypeptide of any one of the following a to c or a variant or an active fragment thereof having an esterase activity and a polynucleotide encoding the same:

a) a polypeptide encoded by a polynucleotide set forth in SEQ ID NO: 1;

b) a polypeptide encoded by a polynucleotide which hybridizes under stringent conditions with the polynucleotide of SEQ ID NO: 1; And,

c) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.

The present invention also provides a recombinant expression vector comprising the polynucleotide.

The present invention also provides a transformant in which the recombinant expression vector is transfected into a host cell.

In addition, 1) preparing a recombinant expression vector comprising a polynucleotide encoding the esterase and its variants or active fragments thereof;

2) preparing a transformant by introducing the recombinant expression vector into a host cell; And,

3) culturing the transformant to induce the expression of a recombinant protein and to provide a method for producing a recombinant esterase consisting of the same.

Hereinafter, the term used by this invention is demonstrated.

In the present invention, a "recombinant expression vector" is a vector capable of expressing a target protein or RNA of interest in a suitable host cell, and is composed of a fragment encoding a polypeptide of interest operably linked to an additional fragment provided for transcription of the expression vector. It is a linear or circular DNA molecule. Such additional fragments include promoter and termination code sequences. Expression vectors also include one or more replication initiation points, one or more selection markers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or contain elements of both.

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 protein or RNA of interest to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect expression of the nucleic acid sequence encoding. Operational linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art and the like.

Hereinafter, the present invention will be described in detail.

The present invention provides a polypeptide of any one of the following a or c, or a variant or active fragment thereof, having an esterase activity and a polynucleotide encoding the same:

a) a polypeptide encoded by a polynucleotide set forth in SEQ ID NO: 1;

b) a polypeptide encoded by a polynucleotide which hybridizes under stringent conditions with the polynucleotide of SEQ ID NO: 1; And,

c) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2.

In a specific embodiment of the present invention, to discover novel esterases exhibiting excellent activity, tributyrin is used in a metagenomic library prepared by extracting DNA from a mud flat soil sample containing microorganisms that cannot be cultured. Colonies that exhibited good esterase activity that effectively degraded were searched for. The recombinant plasmid of the colony was isolated and subjected to sequencing. The 340 amino acids [GenPept Accession No. ACB11219 (1,020 bases; SEQ ID NO: 1, GenBank Accession No. EU515238)] described in SEQ ID NO: 2 consisted of a molecular weight of about 37. An open reading frame (ORF) of the esterase predicted by kDa was found (see FIG. 1). The amino acid sequence of the esterase was compared with the amino acid sequence in GenBank and SWISSPROT using the BLAST program. As a result, Xyella pastiosa 9a5c ( Xylella fastidiosa 9a5c), which showed 37% to 48% homology with the previously reported population of esterase genes and was identified as a novel gene encoding esterase (see Table 1 and Figure 3). ). In addition, the esterase of the present invention is the 186th serine residue (Ser) and the 283th aspartic acid (Asp) in the consensus sequence of Gly-X-Ser-X-Glycine (Gly-X-Ser-X-Gly). ), It had a specific catalytic triad preserved in the esterase consisting of 313th histidine (His) (see FIG. 2).

In a specific embodiment of the present invention, to measure the activity of the novel esterase described in SEQ ID NO: 2, first, the esterase gene of SEQ ID NO: 1 encoding the novel esterase is cloned into a vector (FIG. 4). After the vector was transformed into Escherichia coli, the esterase produced from the transformant was purified. By confirming the purified esterase of the present invention by the SDS-PAGE method, it can be seen that the molecular weight was effectively produced at about 37 kDa, and it was confirmed that the esterase of the present invention was expressed in a water-soluble form ( 5). As described above, since the tidal metagenome-derived esterase of the present invention exhibits excellent activity and is easy to produce, the esterase protein may be widely used as a biocatalyst in biomedical and fine chemical fields as well as in the conversion of fats and oils. .

In addition, the esterases of the present invention exhibit the following properties: stable in the pH range from 6.0 to 11.0, showing optimal activity at pH 8.0 (see FIG. 6); Activity up to 70 ° C. and optimal activity at 50 ° C. (see FIG. 7); Mostly increased activity by divalent metal ions (see Table 1); The activity was increased only for 0.1% of nonionic surfactant, and the activity was rapidly inhibited for the remaining surfactants (see Table 2). In addition, among the various para-nitrophenyl esters (p-nitrophenyl caproate (C6) was the highest activity, which is the para-nitrophenyl caprate (p-nitrophenyl caprate, C10) Compared with), the activity was increased about 13 times, indicating that the protein of the present invention, which degrades a substrate having a relatively short acyl chain, is an esterase rather than a lipase (see FIG. 8). That is, the esterase of the present invention has an activity temperature of 10 to 70 ° C, preferably 50 ° C, and exhibits optimum activity at a relatively high temperature compared to other esterases.

Here, “polynucleotide hybridized under strict conditions” refers to colony hybridization using all or part of a polynucleotide of the nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1 or a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 as a probe. Nucleotide sequences, such as DNA, obtained by techniques, plaque hybridization techniques, southern hybridization techniques, and the like. The hybridization method can be, for example, the method described in Molecular Cloning 3rd Edition, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997 and the like.

As used herein, the term "strict conditions" can be arbitrarily selected from among low stringency conditions, medium stringency conditions or high stringency conditions. "Low stringency conditions" are, for example, 5 x SSC, 5 x Denhardt solution, 0.5% SDS, 50% formamide at 32 ° C. "Medium stringency conditions" are, for example, 5 x SSC, 5 x Denhardt solution, 0.5% SDS, 50% formamide at 42 ° C. "High stringency conditions" are, for example, 5 x SSC, 5 x Denhardt solution, 0.5% SDS, 50% formamide at 50 ° C. Under these conditions, polynucleotides such as DNA, which have higher homology, are expected to be efficiently obtained at higher temperatures, but hybridization stringency includes temperature, probe concentration, probe length, ionic strength, time, salt concentration, and Since a number of factors are involved, including others, those skilled in the art can appropriately select these factors to implement similar stringency.

Polypeptides having an amino acid sequence set forth in SEQ ID NO: 2 of the present invention may be used to identify 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 includes a polypeptide having an amino acid sequence substantially identical to a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, and a variant or active fragment thereof, wherein the substantially identical polypeptide is at least 80%, preferably 90 By those having homology of at least%, most preferably at least 95%, amino acid sequences. However, the present invention is not limited thereto and is included in the present invention as long as it has homology of 70% or more of amino acid sequences and the same biochemical activity.

In addition, the polynucleotide of the present invention is preferably characterized in that the polynucleotide described in SEQ ID NO: 1. However, polynucleotides encoding esterases and variants or active fragments thereof of the present invention are preferred because of degeneracy of codons or in organisms that wish to express the esterases and variants or active fragments thereof. In consideration of the codons, various modifications can be made to the coding region within the range of not changing the amino acid sequence of the esterase expressed from the coding region and the variants thereof or the active fragments thereof, and expression of the gene even in parts other than the coding region. Various modifications or modifications may be made without departing from the scope of the invention, and those skilled in the art will readily understand that such modifications are included in the scope of the present invention. Accordingly, the present invention includes polynucleotides having fragments of the same base and substantially the same nucleotide sequence as the gene of SEQ ID NO: 1. 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%. However, it is not limited thereto and is included in the present invention as long as it has at least 70% sequence homology and the same biochemical activity of the encoded protein. As noted above, one or more nucleic acid bases may be mutated by substitution, deletion, insertion or combination thereof, as long as the polynucleotide of the present invention encodes a protein having equivalent activity, and these also fall within the scope of the present invention. Included. Polypeptides having an amino acid sequence set forth in SEQ ID NO: 2 are preferably encoded by a nucleic acid molecule having a polynucleotide sequence set forth in SEQ ID NO: 1, but the present invention is not limited thereto, and the protein of the present invention having the same amino acid sequence. As long as it can encode, it may be encoded by a nucleic acid molecule having a nucleotide sequence having another base sequence 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.

The present invention also provides a recombinant expression vector comprising an esterase of the present invention and a variant thereof or an active fragment thereof and a polynucleotide encoding the same.

In the production of the recombinant expression vector, expression control sequences such as promoters, terminators, enhancers, etc., depending on the type of host cell intended to produce the esterase and its variants or active fragments thereof. In addition, the sequence for membrane targeting or secretion can be appropriately selected and various combinations can be made depending on the purpose.

Expression vectors of the present 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 expression vector may be constitutive or inducible. The signal sequence includes a PhoA signal sequence and an OmpA signal sequence when the host is Escherichia sp., And an α-amylase signal sequence and a subtilisin signal when the host is Bacillus sp. For example, the MFα signal sequence, the SUC2 signal sequence, and the like may be used when the host is a yeast, and the insulin signal sequence, the α-interferon signal sequence, the antibody molecular signal sequence, or the like may be used when the host is an animal cell. This is not restrictive. 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. Since the expression vector of the present invention expresses its activity when the esterase, a protein of interest, is expressed in the host cell, it is transformed without adding a selection marker by adding an esterase substrate such as tributyrin to the culture medium of the host cell. Selection of host cells can be enabled.

In addition, the recombinant expression vector may include a sequence for facilitating purification of the expression, and specifically, the tag for separation and purification to be operable to the gene encoding the esterase and its variants or active fragments of the present invention. The polynucleotides encoding may be linked. In this case, the separation and purification tag may be used alone, GST, poly-Arg, FLAG, histidine-tag (His-tag) and c-myc, or any two or more of them in sequence. In a specific embodiment of the present invention, the histidine-tag was placed at the C-terminus to purify the expressed esterase using a nickel-entai (Ni-NTA, Ni-nitriloteiacetic acid, Qiagen, Germany) column.

Furthermore, a polynucleotide encoding a protein cleavage enzyme recognition site may be linked to a gene encoding an esterase and a variant thereof or an active fragment thereof of the recombinant expression vector of the present invention and a polynucleotide encoding a tag for separation and purification. Esterases and variants or active fragments thereof of the present invention are proteins that are usefully used as biocatalysts in the fields of biomedical sciences and fine chemistry. In this case, the protein cleavage enzyme recognition site may be used as the Xa factor recognition site, the enterokinase recognition site, the Genenase I recognition site, or the Furin recognition site, or any two or more of them are sequentially connected. have.

In an embodiment of the invention, the gene encoding the esterase and its variants or active fragments thereof can be cloned through a restriction site, and if a polynucleotide encoding a protein cleavage site is used, the polynucleotide Connected in frame so that, when secreted by the proteinase after secreting the esterase and its variants or active fragments, the original form of the esterase and its variants or active fragments may be produced. can do.

The present invention also provides a transformant in which a recombinant expression vector comprising an esterase of the present invention and a variant thereof or an active fragment thereof and a polynucleotide encoding the same is introduced into a host cell.

The recombinant expression vector according to the present invention may be transformed into an appropriate host cell, for example, Escherichia coli or yeast cell, and then cultured in a transformed host cell, thereby producing a large amount of the novel esterase according to the present invention. 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 E. coli, and in a specific embodiment of the present invention, a recombinant expression vector comprising a polynucleotide encoding a novel esterase was prepared (see FIG. 4), and transformed into E. coli. Esterase expressed from the transformant was confirmed to exhibit an excellent esterase activity by effectively decomposing the tributyrin, and the transformant to the biological resource center of the Korea Research Institute of Bioscience and Biotechnology on March 5, 2008 Deposited with accession number KCTC 11285BP.

In addition, 1) preparing a recombinant expression vector comprising a polynucleotide encoding the esterase and variants thereof or active fragments thereof of the present invention;

2) preparing a transformant by introducing the recombinant expression vector into a host cell; And,

3) culturing the transformant to induce the expression of a recombinant protein and to provide a method for producing a recombinant esterase consisting of the same.

The N-terminus of the polynucleotide encoding the esterase of the present invention and the variant or the active fragment thereof may further be linked to the polynucleotide encoding the separation and purification tag and the protein cleavage enzyme recognition site. It may be possible to purify terases or to obtain esterases in their original form. In other words, after the purification of the recombinant esterase using the separation and purification tag, and further comprising the step of processing a protein cleavage enzyme that can cleave the protein cleavage recognition site by obtaining an esterase of the original form Can be.

The esterases of the present invention have unique characteristics such as substrate specificity and optical activity specificity, and thus their use has been attracting attention in developing various bioactive chiral pharmaceutical products. The production of chiral compounds useful for the production of such optically active drugs is attracting attention as a task that has a great influence on securing the competitiveness of the pharmaceutical industry and promoting the health of the public. In particular, the fine chemicals may be drugs of physiologically active chiral drugs, and various techniques such as chemical modification of enzymes, enzyme purification, organic solvent treatment, reaction condition selection, reaction solvent design, etc. may be applied to increase optical activity of enzymes. have.

As described above, the novel esterase produced by the method of the present invention is expressed in water-soluble form, so that it can be mass-produced, maintain activity at high temperature, and exhibit excellent esterase activity to be applied to various industries. Can be.

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  1> Metagenome  Library building

Ten grams of soil samples collected from the Tidal Flats of Buangum Saemangeum Reclaimed Land in Jeollabuk-do were buffered to extract DNA of the same volume containing 50 μg / ml proteinase K [100 mM Trisma buffer solution (Tris-HCl, pH 8.0). , sigma, USA); 100 mM EDTA (ethylenediaminetetraacetic acid, sigma, USA); 100 mM sodium phosphate buffer (sodium phosphate, pH 8.0, sigma, USA); 1.5 M sodium chloride (NaCl, junsei, japan); Suspended in 1% (w / v) CTAB (hexadecyl trimethyl ammonium bromide, sigma, USA), and then added an anionic surfactant (sodium dodecyl sulfate, SDS, sigma, USA) to a final 2% (v / v) The reaction was carried out at 65 ° C. for 2 hours. Thereafter, 30% (v / v) polyethylene glycol including 1.6 M sodium chloride (NaCl) was added to the supernatant obtained through centrifugation, and the mixture was mixed well. The precipitated DNA was separated by centrifugation and suspended in TE buffer, and the same volume of phenol / chloroform / isoamyl alcohol (25: 24: 1) mixture and chloroform / isoamyl alcohol ( Chloroform / isoamyl alcohol) (24: 1) mixture was added and extracted twice. DNA was precipitated by adding isopropanol to the supernatant obtained by centrifugation. Completely dry the DNA precipitate, dissolve in sterile water, completely remove impurities, and electrophoresis using pulse-field gel electrophoresis (Bio-Rad, USA) to partially cut into DNA fragments of about 23 to 48 kb. Gel elution was performed using Gelase (Epicentre, USA). Purified DNA fragments were constructed with a metagenomic library using CopyControl fosmid library construction kit (Epicentre, USA).

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

< Example  2> Esterase  Screening and Isolation of Recombinant Plasmids with Activity

In order to search for an esterase gene from the metagenome library obtained in Example 1, incubating the metagenomic library in a medium containing tributyrin (TBN) degraded by esterase It was.

Specifically, the metagenomic library was prepared using TBN emulsion [1% (v / v) TBN, sigma, USA; 1 mM calcium chloride (CaCl ₂ ), junsei, japan; Nutritional solid medium [1% (w / v) trypton, difco, USA; added 0.5% (v / v) gum arabic, sigma, USA; 0.5% (w / v) yeast extract, difco, USA; 0.5% (w / v) sodium chloride (NaCl), junsei, japan; 1.5% (w / v) agar (agar), junsei, Japan] was incubated at 37 ℃. At this time, colonies forming tricyclic ring by esterase to form a transparent ring were selected. Recombinant plasmids were isolated from colonies with the highest tributyrin resolution and named pFosEstEH112.

< Example  3> new Esterase  Gene Cloning  And sequencing

The gene of the novel esterase was cloned from the plasmid pFosEstEH112 isolated in Example 2 and the nucleotide sequence was analyzed.

<3-1> new Esterase  Gene Cloning

Specifically, plasmid pFosEstEH112 was Sau3AI After digestion with restriction enzymes, E. coli was inserted into a pUC19 vector (Novagen, USA) treated with calf intestinal phosphatase (TaKaRa, Japan). DH5α was transformed. The transformed strain was added to TBN solid medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 1% TBN, 1 mM CaCl ₂ , 0.5% Arabian gum, 1.5% agar) to which ampicillin was added. The colonies were plated and incubated at 37 ° C. for 24 hours to decompose TBN to form transparent rings. As a result of culturing selected colonies to isolate the plasmid, a 4.2 kb plasmid containing 1.5 kb insert DNA was identified, which was named pUC112. In addition, it was confirmed that the colonies generated as a result of re-transforming pUC112 in E. coli DH5α in the same manner all showed an esterase activity.

<3-2> new Esterase  Sequence analysis of genes

The base sequence of pUC112 obtained in Example 3-1 was analyzed using a Big Dye DNA Sequencing kit (AppliedBiosystems, USA) by shot-gun sequencing. The base sequence was identified by using an ORF finder (//www.ncbi.nlm.nih.gov/gorf/gorf.html) of the National Center for Biotechnology Information (NCBI) to identify an open reading frame (ORF). , BlastX (www.ncbi.nlm.nih.gov/blast/Blast.cgi) was used to predict the function of the transcription decoding frame.

As a result, an open reading frame (ORF) of esterase consisting of 340 amino acids (SEQ ID NO: 1; 1,020 bases) described in SEQ ID NO: 2 and predicted to have a molecular weight of about 37 kDa was found (FIG. 1). . The ORF is expected to be an esterase protein coding region, and the gene was named esterase EstEH112 and registered in NCBI (GenPept Accession No. ACB11219; GenBank Accession No. EU515238).

<3-3> new Esterase  Gene Homology  analysis

The amino acid sequence of the novel esterase of SEQ ID NO: 2 was compared with the amino acid sequences in GenBank and SWISSPROT using the BLAST program.

As a result, the esterase of the present invention EstEH112 is Zyella Pastidiosa 9a5c ( Xylella fastidiosa 9a5c), which showed 37% to 48% homology with the previously reported population of esterase genes and was identified as a novel gene encoding esterase (see Table 1 and Figure 3). ). Also, as shown in FIG. 2, the esterase of the present invention is the 186th serine residue in the consensus sequence of glycine-X-serine-X-glycine (Gly-X-Ser-X-Gly). And 283 aspartic acid (Asp) and 313 histidine (His). In addition, marine gamma proteobacterium HTCC2143 ( Zylella Pastiosa 9a5c ( Xylella ) similar to EstEH112 as shown in Table 1 and Figure 3 fastidiosa 9a5c), methyl tumefaciens species 4-46 (Methylobacterium sp. 4-46), the cine nose Im seutiseu species PCC 6803 (Synechocystis sp. PCC 6803 ), a Pseudomonas Alter do nucleoside D 'deep ecotype' (Alteromonas macleodii 'Deep ecotype'), a bacterium methyl Solarium nodul lance ORS 2060 (Methylobacterium nodulans ORS 2060), Granulibacter Bethesdensis CGDNIH1 ( Granulibacter Esterase enzymes of bethesdensis CGDNIH1) are genes that have recently been identified by whole genome sequencing, and enzymes have never been characterized.

Proteins similar to EstEH112 Protein name Microbial sources GenBank  number Equal Amino Acid Ratio / Similar Amino Acid Ratio (%) E- value Esteh112 Metagenome EU515238 - - esterase marine gamma proteobacterium HTCC2143 EAW33037 46/66 4e-85 esterase Xylella fastidiosa 9a5c AAF84552 43/61 3e-70 Alpha / beta hydrolase fold-3 domain protein Methylobacterium sp. 4-46 ACA15557 42/61 3e-64 esterase Synechocystis sp. PCC 6803 BAA16971 38/58 3e-61 esterase Alteromonas macleodii 'Deep ecotype' EAR05306 34/56 6e-54 esterase Methylobacterium nodulans ORS 2060 EDQ46384 37/53 1e-50 esterase Granulibacter bethesdensis CGDNIH1 ABI62669 33/50 1e-40

< Example  4> Preparation of transformant

Recombinant plasmids containing the EstEH112 gene capable of producing large amounts of the novel esterases of the present invention were prepared (FIG. 4).

Specifically, pFosEstEH112 isolated by the method of Example 2 was used as template DNA, and the synthesized SEQ ID Nos. 3 and 4 are described as Nde I and Polymerase chain reaction (PCR) was performed using a primer pair having a restriction enzyme cleavage site of Xho I.

SEQ ID NO: 5 '-CCGCTCGAGTTGTAGGTGCTGCAATAGAA-3' ( Nde I Restriction enzyme cleavage site)

SEQ ID NO: 5'- GGAATTCCATATGATGACAAGCCAAGAATCAAC-3 '( Xho I Restriction enzyme cleavage site)

Purifying the PCR product and Nde I and Restriction enzyme of the expression vector pET-22b (+) (Novagen, USA), which was digested with Xho I restriction enzymes, and had a strong T7 promoter treated with the same restriction enzyme and calf intestinal phosphatase, and a readout signal. Cloning the EstEH112 gene of SEQ ID NO: 1 to the Nde I and Xho I sites (recombinant vector pET22b (+)-EstEH112), and transformed it to E. coli C43 (DE3) by electron shock gene transfer (electroporation) to transform E. coli C43 ( DE3) / pET22b (+)-EstEH112 was prepared, and the strain was deposited on March 5, 2008 at the Bioresource Center of the Korea Research Institute of Bioscience and Biotechnology (Accession No .: KCTC 11285BP).

< Example  5> Esterase  production

<5-1> Esterase  Expression and Purification

Escherichia coli C43 (DE3) / pET22b (+)-EstEH112 obtained in Example 4 was absorbed at 37 ° C in a liquid nutrient medium containing ampicillin (ampicillin, 100 μg / ml) until the absorbance at 600 nm was 0.6. After incubation, the final concentration of 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the culture solution and further incubated at 20 ° C. for 12 hours. E. coli C43 (DE3) / pET22b (+)-EstEH112 was recovered by centrifugation, and bound to binding buffer [50 mM Trisma buffer (Tris-HCl, pH 8.0), 500 mM NaCl, 10 mM imidazole]. After suspension, the mixture was broken by ultrasonic grinding. Only the supernatant recovered by centrifugation was added to a nickel-NTA (ni-NTA: nitriloteiacetic acid; Qiagen, Germany) column, and the esterase was eluted using an imidazole concentration gradient.

<5-2> Esterase  Confirm

The protein concentration purified in Example 5-1 was measured by absorbance at 280 nm using a NanoDrop Spectrophotometer (nanodrop, USA), and then 5 × SDS (0.156 M Tris-HCl, pH 6.8, 2.5). % SDS, 37.5% glycerol, 37.5 mM DTT) 1: 1 and mixed and boiled for 10 minutes at 100 ℃. Boiled samples were loaded into wells of a 10% SDS-PAGE gel, the samples were developed at 125 V for 2 hours, and the gels were stained by Coomassie staining and then decolorized to confirm the expression level of each recombinant protein.

As a result, as shown in Figure 5, the esterase of the present invention was found that the molecular weight of the protein in the sample was effectively produced at about 37 kDa after 12 hours of expression, which is quite similar to the molecular weight inferred from the amino acid sequence of the esterase. In close proximity, it was confirmed that this protein band is the novel esterase of the present invention. It was also confirmed that the esterase of the present invention was expressed in water-soluble form.

< Example  6> tidal flat From metagenome  Separated Esterase  Characteristic investigation

The enzyme activity, specificity for substrates with various carbon lengths, and effects on various metal ions and surfactants of the esterase EstEH112 purified in Example 5-1 were measured. Specifically, the reaction solution for measuring enzyme activity was 10 μl of 10 mM p-nitrophenyl ester substrate, 40 μl of ethanol, 940 μl of 50 mM Trisma buffer (Tris-HCl, pH 8.0), and 5 µl or 60 minutes of reaction was performed by adding 5 µl of the esterase enzyme purified in Example 5-1, and the para-nitrophenol released from the substrate was measured at an absorbance increase rate at 405 nm. At this time, one unit (U) was defined as the amount of enzyme that can be produced by hydrolyzing 1 μmol para-nitrophenol per minute.

<6-1> pH For Esterase  characteristic

In order to investigate the effect of pH on the esterase activity, the activity was measured for 5 minutes in various reaction solutions of pH 4 to 12 showed the maximum activity at pH 8.0 (Fig. 6a), 60 minutes at the various pH After standing, the residual activity of the esterase was measured and found to be stable in a wide range of pH 6.0-11.0 (FIG. 6B).

<6-2> for temperature Esterase  characteristic

As a result of investigating the effect of temperature on the esterase activity, the activity was measured for 5 minutes in the reaction solution at various temperatures of 10 to 70 ℃ showed the optimum activity at 50 ℃ (Fig. 7a), to investigate the stability to heat After remaining for 60 minutes at each temperature, the residual enzyme activity was measured and found to be stable as the activity was maintained in the temperature range up to 50 ° C (FIG. 7B).

<6-3> for various carbon lengths Esterase  characteristic

In order to determine the specificity for the substrates having various carbon lengths, the para-nitrophenyl ester of the reaction solution was selected from C2 to C18 [para-nitrophenyl acetate (C2), para-nitrophenyl propionate p. -nitrophenyl propionate (C3), para-nitrophenyl butyrate (C4), para-nitrophenyl caproate (C6), para-nitrophenyl caprylate (p-nitrophenyl caprylate, C8), para-nitrophenyl caprate (p10), para-nitrophenyl laurate (p12), para-nitrophenyl myristate (p14), para- Esterase activity was compared in the same way by substituting substrates of nitrophenyl palmitate (C16) and para-nitrophenyl stearate (C18)].

As a result, as shown in Figure 8 among the various para-nitrophenyl ester (p-nitrophenyl ester) was the highest activity for p-nitrophenyl caproate (p-nitrophenyl caproate, C6), which is para-nitrophenyl cap Compared to the rate (p-nitrophenyl caprate, C10), the activity was increased by about 13 times. Therefore, it can be seen that EstEH112, which better decomposes substrates with relatively short acyl chains, is esterase, not lipase.

<6-4> for metal ions Esterase  effect

As a result of measuring the activity of the esterase for various metal ions of various concentrations as shown in Table 2 below, it was confirmed that the activity is similar or insignificantly increased by most divalent metal ions.

Enzyme Activity of Esterases on Metal Ions Metal ion Enzyme activity relative value (%) manufacturer 1 mM 5 mM 10 mM Calcium chloride (CaCl ₂ ) 102 99 90 junsei, japan Copper Sulfate (CuSO ₄ ) 97 90 61 junsei, japan Nickel Sulfate (NiSO ₄ ) 102 99 88 junsei, japan Zinc Sulfate (ZnSO ₄ ) 96 94 87 junsei, japan Manganese Sulfate (MnSO ₄ ) 101 97 91 junsei, japan Cobalt Chloride (CoCl ₂ ) 102 101 88 junsei, japan Magnesium Sulfate (MgSO ₄ ) 102 97 92 junsei, japan Ferrous Sulfate (FeSO ₄ ) 96 39 9 junsei, japan

<6-5> for surfactants Esterase  effect

As a result of measuring the activity of the esterase for various surfactants of various concentrations as shown in Table 3 below, the activity was increased only for the nonionic surfactant of 0.1%, and the activity was rapidly inhibited for the remaining surfactants. Confirmed.

Enzyme Activity of Esterases on Surfactants Surfactants Enzyme activity relative value (%) manufacturer 0.1% One% Tween 20 104 43 junsei, japan Tween 40 114 19 junsei, japan Tween 60 113 0 junsei, japan Tween 80 21 0 junsei, japan Triton X-100 8 0 sigma, USA Na-taurocholate 0 0 sigma, USA Deoxycholate 0 0 sigma, USA CHAPS 0 0 sigma, USA

While the present invention has been described in detail through practice, it will be understood by those skilled in the art that these specific techniques are merely preferred embodiments and thus are not intended to limit the scope of the present invention.

1 is a diagram showing the nucleotide sequence and amino acid sequence of the esterase EstEH112 of the present invention (*: stop codon).

Figure 2 shows the result of comparing the homology between the amino acid sequence of the esterase EstEH112 of the present invention and the amino acid sequence of the similar esterase. The amino acids marked with ▼ indicate a well-conserved esterase specific catalytic triad.

Figure 3 is a comparative analysis of the nucleotide sequence of the various esterases having high homology with the esterase EstEH112 of the present invention.

4 is a schematic diagram of a recombinant vector pET22b (+)-EstEH112 comprising a novel esterase gene derived from a tidal flat metagenomic according to the present invention.

5 is a diagram showing the results of confirming the purification of the desired 37 kDa esterase in E. coli transformed with the tidal metagenome-derived esterase gene by SDS-PAGE:

Lane 1: molecular weight standard marker; And,

Lane 2: final pure purified esterase through a Ni-NTA column.

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

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

8 is a graph showing the degradation activity of the substrate having various carbon lengths of the esterase.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel gene encoding esterase derived from tidal flat metagenome          and preparation method <130> 8P-03-46 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1020 <212> DNA <213> Unknown <220> <223> uncultured bacterium <400> 1 atgacaagcc aagaatcaac taccggaaat aaatcccgag aaacagtctg gcggatcggt 60 ccccgaatcc ttccgccacc gactggcggc agcgctgaac tgcgtgacgc tattgccagc 120 atcccccagc ctgatccggc aacgatgaaa atcgagcctc agagcgagga agagtggctc 180 gcggtcatcg cgcagctgga cgcgggcaag gtcgaagcca atcaggccct cagggaacag 240 ctatcggttt ccgttacaca agaagagatt gaggggatca atgtctatca tttgacccca 300 gccgaagtcg atccccggca cgaggatcat ttgttcgtct acctgcatgg cggcgccttc 360 gtgctcaacg ctggcgaagc gggtttagca gaatctatca tcatcgccca ccggctgaag 420 atgcggctga tgcatattga ctaccgcatg ccgcccaaat atcctacccc ggccggaagg 480 gacgatgtag tcaccgtgta caagcacctg ttgaaagaaa gaccggcgcg ttccatggcg 540 atgggcggca cctcaggagg cgcgaatatg accatggcga cggtgcagcg cttgattgag 600 ctgggcctgg atgtgcctgg ggtgttgtac ctgggcacac ctgggactga tgtgtccaac 660 acgggtgact cctggattat caacgagggc attgaccata tattgatcac ccgcgagggc 720 atgctcgaag cttgtgtggc cgtgtacgcg gctggccgcg atccgaaaga tcccttggtc 780 tcaccttact atggagatgt ccatggattt ccacccacct tgttgataac gggaacgcgg 840 gacatgctct tgagcagcac cgctcgcacc catatcaaac tgcgtcaagc gggagttgta 900 gcagacattc tggtctacga tggtgtttcc catggagatt acatcttcgt aatgaacgcg 960 ccggagtcag cgcatgctta cgctgagctc aacgcgtttc tattgcagca cctacaataa 1020                                                                         1020 <210> 2 <211> 339 <212> PRT <213> Unknown <220> <223> uncultured bacterium <400> 2 Met Thr Ser Gln Glu Ser Thr Thr Gly Asn Lys Ser Arg Glu Thr Val   1 5 10 15 Trp Arg Ile Gly Pro Arg Ile Leu Pro Pro Thr Gly Gly Ser Ala              20 25 30 Glu Leu Arg Asp Ala Ile Ala Ser Ile Pro Gln Pro Asp Pro Ala Thr          35 40 45 Met Lys Ile Glu Pro Gln Ser Glu Glu Glu Trp Leu Ala Val Ile Ala      50 55 60 Gln Leu Asp Ala Gly Lys Val Glu Ala Asn Gln Ala Leu Arg Glu Gln  65 70 75 80 Leu Ser Val Ser Val Thr Gln Glu Glu Ile Glu Gly Ile Asn Val Tyr                  85 90 95 His Leu Thr Pro Ala Glu Val Asp Pro Arg His Glu Asp His Leu Phe             100 105 110 Val Tyr Leu His Gly Gly Ala Phe Val Leu Asn Ala Gly Glu Ala Gly         115 120 125 Leu Ala Glu Ser Ile Ile Ile Ala His Arg Leu Lys Met Arg Leu Met     130 135 140 His Ile Asp Tyr Arg Met Pro Pro Lys Tyr Pro Thr Pro Ala Gly Arg 145 150 155 160 Asp Asp Val Val Thr Val Tyr Lys His Leu Leu Lys Glu Arg Pro Ala                 165 170 175 Arg Ser Met Ala Met Gly Gly Thr Ser Gly Gly Ala Asn Met Thr Met             180 185 190 Ala Thr Val Gln Arg Leu Ile Glu Leu Gly Leu Asp Val Pro Gly Val         195 200 205 Leu Tyr Leu Gly Thr Pro Gly Thr Asp Val Ser Asn Thr Gly Asp Ser     210 215 220 Trp Ile Ile Asn Glu Gly Ile Asp His Ile Leu Ile Thr Arg Glu Gly 225 230 235 240 Met Leu Glu Ala Cys Val Ala Val Tyr Ala Ala Gly Arg Asp Pro Lys                 245 250 255 Asp Pro Leu Val Ser Pro Tyr Tyr Gly Asp Val His Gly Phe Pro Pro             260 265 270 Thr Leu Leu Ile Thr Gly Thr Arg Asp Met Leu Leu Ser Ser Thr Ala         275 280 285 Arg Thr His Ile Lys Leu Arg Gln Ala Gly Val Val Ala Asp Ile Leu     290 295 300 Val Tyr Asp Gly Val Ser His Gly Asp Tyr Ile Phe Val Met Asn Ala 305 310 315 320 Pro Glu Ser Ala His Ala Tyr Ala Glu Leu Asn Ala Phe Leu Leu Gln                 325 330 335 His Leu Gln             <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 3 ccgctcgagt tgtaggtgct gcaatagaa 29 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 4 ggaattccat atgatgacaa gccaagaatc aac 33  

Claims (10)

A polypeptide of any one of the following a or c or a variant or active fragment thereof having esterase activity: a) a polypeptide encoded by a polynucleotide set forth in SEQ ID NO: 1; b) a polypeptide encoded by a polynucleotide which hybridizes under stringent conditions with the polynucleotide of SEQ ID NO: 1; And, c) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2. The esterase and its variants or active fragments thereof according to claim 1, wherein the esterase has an active temperature of 10 to 70 ° C. A polynucleotide encoding the esterase of claim 1 and variants or active fragments thereof. The polynucleotide of claim 3, wherein the polynucleotide is set forth in SEQ ID NO: 1. Recombinant expression vector comprising the polynucleotide of claim 3. A transformant transformed with a recombinant expression vector of claim 5 into a host cell. The transformant according to claim 6, wherein the transformant has been deposited with accession number KCTC 11285BP. 1) preparing a recombinant expression vector comprising the polynucleotide of claim 3; 2) preparing a transformant by introducing the recombinant expression vector into a host cell; And, 3) culturing the transformant to induce the expression of a recombinant protein and to obtain a method for producing a recombinant esterase. The method according to claim 8, wherein the polynucleotide encoding the separation and purification tag and the protein cleavage enzyme recognition site are further connected to the N-terminus of the polynucleotide of step 1). The method according to claim 8, further comprising the step of treating the recombinant esterase with a protease capable of cleaving the proteolytic enzyme recognition site of claim 9 to obtain an esterase in its original form. Way.

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