KR101715817B1 - Novel lipolytic enzyme from metagenomic library and process for preparing the same - Google Patents

Abstract

The present invention relates to novel esterase (Est3K) or lipase (Lip3K) derived from a metagenomic library; a polynucleotide for coding the esterase (Est3K) or the lipase (Lip3K); a recombinant vector including the polynucleotide; a transformant having the recombinant vector introduced thereto; and a method for producing esterase (Est3K) or lipase (Lip3K) by using a cell having a polynucleotide for coding the transformant, esterase, or lipase. The esterase (Est3K) and the lipase (Lip3K) have alkali properties and heat resistance, and each thereof shows an activity of selectively hydrolyzing p-nitropenyl butyrate (C4) and p-nitropenyl octanoate (C8). In particular, the lipase (Lip3K) has an improved lipolytic activity under the presence of Ca2+ ions or 30% methanol, thereby being able to be used not only for a milk fat decomposing agent, an agent for decomposing lipid in milk products, a fodder composition, a cleansing composition, a fiber product additive, or a molecular synthetic additive composition but also for a strong biocatalyst of an ester exchange reaction for producing biodiesel.

Description

TECHNICAL FIELD The present invention relates to a novel lipolytic enzyme derived from a metagenomic library,

The present invention relates to a novel lipolytic enzyme derived from a meta genome library and a production method thereof, and more particularly to a novel lipolytic enzyme derived from a meta genome library, a polynucleotide encoding said lipolytic enzyme, A transformant into which the recombinant vector is introduced, and a method for producing a lipolytic enzyme using the transformant or a cell having a polynucleotide encoding the lipolytic enzyme.

Lipolytic enzymes, esterases and lipases, are carboxylic ester hydrolases that promote the hydrolysis of glycerol and are capable of hydrolyzing α / β hydrolase (α / β hydrolase (Ollis, DL, et al. , 1992, Protein Engineering, 5: 197-211). Among the lipolytic enzymes, esterase hydrolyzes ester bonds of short chain fatty acids containing less than 10 carbons, and lipase hydrolyzes ester bonds of 10 or more long chain fatty acids Disassemble. Such esterases and lipases are widely used for the synthesis and production of biopolymers, biodiesel, chiral building blocks, pharmaceuticals, aromatic compounds and pesticides as well as for the decomposition of racemic mixtures (Jaeger, KE, and Eggert , T., 2002, Current Opinion in Biotechnology, 13: 390-397).

Lipolytic enzymes have been found in various animals, plants, fungi and bacteria. Among them, lipolytic enzymes produced by microorganisms (fungi and bacteria) have various substrate specificity, site specificity and stereospecificity, and thus they are widely used industrially.

Bacterial lipolytic enzymes are divided into 8 families and recently, new lipolytic enzymes have been discovered and expanded to 15 groups (Arpigny, KE and Jaeger, KE, 1999, Biochemical Journal, 343: 177-183. ; Charbonneau, DM and Beauregard, M., 2013, PLoS One, 8: e76675.). Specifically, Family I is subdivided into seven subgroups (I.1 to I.7), including the subfamilies I.1, I.2, and I.3, which are the largest groups isolated from gram negative bacteria (Jaeger , KE, and Eggert, T., 2002, Current Opinion in Biotechnology, 13: 390-397.). The structure and function of the associative I.3 enzyme is compared to the alleged I.1 and I.2 enzymes, typically the host I.3 lipase is secreted through a Type I secretion system. Family IV lipase is a group of hormone sensitive lipases (HSL) whose amino acid sequence is similar to that of mammals (Arpigny, K. E. and Jaeger, K. E., 1999, Biochemical Journal, 343: 177-183).

Metagenomes, on the other hand, are environmental samples that constitute the genome of many individuals and are associated with all genetic material (Handelsman, J. 2004, Microbiology and Molecular Biology Reviews, 68: 669-685. ). Such metagenomes are useful for the detection of new biocatalysts, and have the advantage of studying microbial genes without isolating or culturing microorganisms. Many esterase and lipase genes have been reported to be able to separate from metagenomes in a variety of environments including marine environments, soil, water, forest soils, and compost (Biver, S. and Vandenbol, M., 2013, Applied Microbiology Chow, J., et al. , 2012, PLoS One, 7: e47665, et al. , 2014, Journal of Microbiology and Biotechnology, 24: 771-780 ; Bunterngsook, B., et al. , 2010, Bioscience, Biotechnology and Biochemistry, 74: 1848-1854 .; Kim, YH, et al. , 2010, Biochemical and Biophysical Research Communications, 393: 45-49.).

Based on the fact that various esterase and lipase genes can be separated from the meta genome library of various environments, the present inventors have isolated and identified novel esterase and lipase from the metagenomic library of oil-contaminated tidal flats, And lipase have alkalinity and heat resistance, and confirmed that the fatty acid is efficiently hydrolyzed, thereby completing the present invention.

One object of the present invention is to provide a novel esterase derived from a meta genome library.

It is another object of the present invention to provide a novel lipase derived from a metagenomic library.

It is another object of the present invention to provide a polynucleotide encoding the Esterase or a lipase.

It is another object of the present invention to provide a recombinant vector comprising the polynucleotide encoding the Esterase or the lipase.

It is still another object of the present invention to provide a transformant transformed by introducing a recombinant vector comprising the polynucleotide encoding the Esterase or the lipase.

It is still another object of the present invention to provide a method for producing an esterase or a lipase using the transformant, or a cell having a polynucleotide encoding the Esterase or a lipase.

In some embodiments, the present invention is a molecular weight of 34 kDa, and the optimum active temperature and pH is 50 ℃ and pH 9.0, respectively, pH 8.0 to 10.0 and 40 to 55 ℃ the enzyme activity has a stable alkaline and heat resistance in a, p - And then hydrolyzing the nitrophenylbutyrate (C4) selectively.

In the present invention, the esterase is encoded by a polynucleotide of 900 bp in total, is translated into 299 amino acids, and does not have a signal peptide.

Also, the Esterase is composed of the amino acid sequence of SEQ ID NO: 2, the 'HGGG' motif is in the 74th to 78th amino acid region, the 'GXSXG' motif is in the 144th to 148th amino acid region, 'Motif in the 244th to 246th amino acid region, and the' HGF 'motif in the 270th to 272nd amino acid regions. This means that the esterase of the present invention belongs to Family IV of lipolytic enzymes.

Therefore, the esterase of the present invention is a novel lipolytic enzyme belonging to Family IV. In the present invention, the esterase having the above-mentioned characteristics is named Est3K.

In the present invention, the novel Esterase can be obtained by introducing a metagenome library into a host cell and purifying the transformant from the transformant. At this time, the metagenome library is obtained by collecting a microorganism community existing in nature or a specific area (hot spring, farm, compost, oil polluted area), directly extracting the genome from the microorganism community, and introducing it into a vector.

Specifically, the esterase of the present invention can be prepared by introducing a metagenomic library into a host cell to prepare a transformant, culturing the transformant, and purifying the transformant from the culture supernatant through a protein purification method known in the art . Herein, the host cell is chemically treated with a normal host cell for transformation, and the foreign gene is able to enter the cell and express the foreign gene. The normal host cell is E. coli , , Bacillus (Bacillus) strain, Salmonella typhimurium (Salmonella typhimurium) strain, Serratia marcescens, or the like, but the sense Marseille (Serratia marcescens) strain and the genus Pseudomonas (Pseudomonas) strain, and the like.

The purification method can be carried out, for example, by purifying an esterase by separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (size, charge, hydrophobicity or affinity) .

The esterase according to the present invention has alkaline and heat resistance and exhibits an activity of hydrolyzing p -nitrophenylbutyrate (C4) at a pH of 8.0 to 10.0 and a temperature of 40 to 55 ° C, so that the lipase in the milk fat cleaver, A feed composition, a detergent composition, a fiber product additive or a composition for synthesizing a polymer.

In another one aspect, this invention provides a molecular weight of 64 kDa, and the optimum active temperature and pH is 50 ℃ and pH 9.0, respectively, pH 7.0 to 9.5 and 45 to 55 ℃ the enzyme activity has a stable alkaline and heat resistance at, p - hydrolysis of nitrophenyl octanoate (C8), the lipolytic activity and the thermal stability were increased by 180 to 200% and 20 to 40%, respectively, when Ca ²⁺ ion was added, and the lipolytic activity Is increased to 150 to 180%.

In the present invention, the lipase is encoded by a polynucleotide of 1,951 bp in total, is translated into 616 amino acids, and does not have a signal peptide.

Also, the lipase is composed of the amino acid sequence of SEQ ID NO: 4, the 'VTLVG' motif is in the 598th to 602th amino acid region, the 'GGXGDXUX' motif is the 372st to 416th amino acid, the 492st to 545th amino acid , And in the 548th to 556th amino acid regions, and the 'GXSXG' motif in the 204th to 208th amino acid regions. This means that the lipase of the present invention belongs to Family I.3 among lipolytic enzymes.

Therefore, the lipase of the present invention is a novel lipolytic enzyme belonging to Family I.3. In the present invention, the lipase having the above-mentioned characteristics was named Lip3K.

The lipase of the present invention can be obtained by introducing a metagenome library into a host cell in the same manner as the above-mentioned Esterase, and purifying the transformant from the transformant.

Specifically, the lipase of the present invention can be prepared by introducing a metagenome library into a host cell to prepare a transformant, culturing the transformant, and purifying the transformant from the culture supernatant through a protein purification method known in the art . The characteristics of the host cell, the kind of the host cell, the method of purification, and the like are the same as those described above, and thus will not be described below.

As it described above, the lipase according to the invention has an alkali and heat resistance, pH 7.0 to 9.5 and p 45 to 55 ℃ - represents the hydrolysis activity of the nitrophenyl octanoate (C8), particularly Ca ²⁺ ions Or 30% methanol, the lipid decomposing activity is improved. Therefore, the lipid decomposition agent or the lipid disintegration agent in the dairy product, the feed composition, the detergent composition, the fiber product additive or the polymer synthesis additive composition as well as the bio- Can be used.

In yet another embodiment, the present invention provides a polynucleotide encoding an esterase (Est3K) or lipase (Lip3K) exhibiting the above-mentioned characteristics. In this case, the polynucleotide encoding the Esterase (Est3K) may have the nucleotide sequence of SEQ ID NO: 1, and the polynucleotide encoding lipase (Lip3K) may have the nucleotide sequence of SEQ ID NO: 3.

As used herein, the term "polynucleotide " is used interchangeably with terms such as a polynucleotide sequence, a polynucleotide molecule, a nucleic acid, a nucleic acid molecule and a nucleic acid sequence. The nucleic acid may be a ribonucleic acid or DNA (deoxyribonucleic acid) .

In yet another embodiment, the present invention provides a recombinant vector comprising a polynucleotide encoding said esterase (Est3K) or lipase (Lip3K).

The term "vector" as used herein refers to a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host cell. The vector may be a plasmid, phage particle, or simply a potential genome insert. Since plasmids among these vectors are the most commonly used forms, the terms "plasmid" and "vector" in the present specification can be used interchangeably.

The vectors that can be used in the present invention include: (a) a cloning start point that allows efficient replication of several hundreds of vectors per host cell; (b) an antibiotic resistance gene that allows selection of a host cell transformed with the vector And (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA.

In another embodiment, the present invention provides a transformant transformed by introducing a recombinant vector comprising a polynucleotide encoding the above-mentioned Esterase (Est3K) or lipase (Lip3K) into a host cell.

In the present invention, the host cell is chemically treated with a normal host cell for transformation so that the foreign gene can enter the cell well and express the foreign gene. The normal host cell is Escherichia coli ( E .coli), Bacillus (Bacillus) strain, Salmonella typhimurium (Salmonella typhimurium) strain, Serratia marcescens, or the like, but the sense Marseille (Serratia marcescens) strain and the genus Pseudomonas (Pseudomonas) strain, and the like.

In the present invention, the method of transforming the recombinant vector into a host cell is not particularly limited as long as the recombinant vector can be inserted into the host cell. For example, the CaCl ₂ method, the electroporation method, or the microinjection method can be used Do.

As used herein, the term "recombinant vector" means a recombinant carrier into which a different kind of DNA fragment is inserted, and generally refers to a double-stranded DNA fragment. Here, the heterologous DNA means a heterologous DNA which is a DNA that is not naturally found in a water-soluble cell. Once the recombinant vector is in a water-soluble cell, it can be replicated independently of the chromosomal DNA of the water-soluble cell, and several copies of the vector and its inserted heterologous DNA can be produced.

As used herein, the term "transfection " is used interchangeably with the introduction, and includes both delivery of the foreign polynucleotide to the host cell, regardless of the method used.

In another aspect, the present invention provides a method for producing an esterase (Est3K) or lipase (Lip3K), which comprises the following steps.

a) culturing a transformant into which a recombinant vector containing a polynucleotide encoding the above-mentioned Esterase (Est3K) or lipase (Lip3K) has been introduced or a cell having a polynucleotide encoding said esterase (Est3K) or lipase (Lip3K) step; b) inducing expression of an esterase (Est3K) or lipase (Lip3K) in the transformant or cells; (c) eluting an esterase (Est3K) or lipase (Lip3K) from a supernatant obtained by pulverizing and then precipitating a transformant or cells expressing said esterase (Est3K) or lipase (Lip3K); And d) purifying the eluted Esterase (Est3K) or lipase (Lip3K).

As the host cell capable of transforming the recombinant vector in step a), any cell can be used as long as the foreign gene can enter the cell well and display the trait of the foreign gene. example, may be used such as Escherichia coli (E.coli), Bacillus (Bacillus) strain, Salmonella typhimurium (Salmonella typhimurium) strain, Serratia Marseille sense (Serratia marcescens) strain and the genus Pseudomonas (Pseudomonas) strains.

In step a), the transformant or cells can be cultured in LB (Luria-Bertani) liquid medium. Preferably, the transformant or cells can be cultured in a medium such as ampicillin, kanamycin and tetra And LB liquid medium containing an antibiotic such as cyclin, more preferably, LB liquid medium containing ampicillin.

In step b), induction of expression of Esterase (Est3K) or lipase (Lip3K) may be induced by treating an inducer such as allolactose and IPTG (Isopropyl-β-D-Thiogalactopyranoside) IPTG can be induced to a final concentration of 0.01 to 1 mM, preferably 0.05 to 0.5 mM, more preferably 0.1 mM.

The elution of the esterase (Est3K) or lipase (Lip3K) in the step c) is eluted using a buffer solution containing a salt of an inorganic acid or an organic acid which does not affect the biological function of the esterase (Est3K) or lipase (Lip3K) . For example, it may be a buffer solution comprising citrate, acetate, acuminate, lactate, tartrate, formate, propionate, phosphate, or borate.

The purification of the esterase (Est3K) or lipase (Lip3K) in the step d) can be carried out using a protein purification method known in the art, preferably by ion exchange chromatography, gel-permeation chromatography, reversed phase-HPLC , SDS-PAGE for prophage, and affinity column chromatography.

Refers to nitrophenyl octanoate (C8) optionally hydrolyzing activity of a-esterase (Est3K) and lipase (Lip3K) is alkaline and has a heat resistance, each of p in accordance with the present invention-nitrophenyl butyrate (C4), and p In particular, lipase (Lip3K) has improved lipolytic activity in the presence of Ca ²⁺ ions or 30% methanol. Therefore, the esterase (Est3K) and lipase (Lip3K) of the present invention are useful as a lipid disintegrator, a lipid disintegrant in dairy products, a feed composition, a detergent composition, a fiber product additive or a polymer synthesis additive composition, It can be used as a biocatalyst and the like, and thus its industrial value will be very great.

FIG. 1 is an alignment chart of the amino acid sequence of Est3K of the present invention and the conventional lipolytic enzyme. FIG.
FIG. 2 is a diagram showing the alignment of the amino acid sequences of Lip3K of the present invention and conventional lipolytic enzymes. FIG.
FIG. 3 is a diagram showing the tree numbers of Est3K and Lip3K of the present invention. FIG.
FIG. 4 is a gel photograph of SDS-PAGE analysis of the molecular weight of Est3K or Lip3K purified through chromatography from SKE3 strain having esterase activity and SKEL5 strain having esterase activity and lipase activity.
FIG. 5 is a graph showing the activity of Est3K or Lip3K of the present invention to decompose p-nitrophenyl ester.
FIG. 6 is a graph showing changes in enzyme activity of Est3K or Lip3K of the present invention according to the reaction temperature. FIG.
7 is a graph showing changes in enzyme activity of Est3K or Lip3K of the present invention depending on reaction temperature and time.
8 is a graph showing changes in enzyme activity of Est3K or Lip3K of the present invention according to pH.
9 is a graph showing changes in enzyme activity of Lip3K of the present invention depending on reaction temperature and time in the presence of Ca ²⁺ ion.

Hereinafter, the present invention will be described in more detail with reference to examples. It will be apparent to those skilled in the art that these embodiments are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1: Construction of a metagenomic library from oil-contaminated tidal flats and selection of an esterase / lipase active clone

Metagenomic DNA of 30 to 60 kb in size was extracted from the tidal flats of the oil-contaminated Taean county using the method described by Kim et al . (Biochem Biophys Res Commun., 2010, 393 (1): 45-49). The extracted meta genomic DNA was treated with Sau 3AI to cut the DNA to a size of 3 to 8 kb, and electrophoresed on 0.7% agarose gel to elute the meta genomic DNA. The eluted metagenomic DNA was then ligated into a pUC19 vector truncated with Bam HI and introduced into E. coli DH5a (Yeastern Biotech. Co., Taipei, Taiwan) using a heat-shock method . E. coli into which the pUC19 vector containing the meta genomic DNA was introduced was plated on LB solid medium containing ampicillin (50 / / ml), X-Gal, and IPTC, and cultured at 37 캜 for 24 hours. Then, the above cultured strains were collected with a toothpick, and then inoculated on LB solid medium containing 1.0% glyceryl tributyrate or 1.0% glyceryl trioctanoate, respectively, and cultured at 37 ° C for 24 hours Lt; RTI ID = 0.0 > Esterase / lipase < / RTI > activity.

As a result, strains having seven esterase activities and strains having two lipase activities were selected. Especially, among the selected strains, the SKE3 strain showed high esterase activity, and SKEL5 showed the activity of esterase and lipase.

Example 2 Analysis of Gene and Amino Acid Sequence of Esterase and / or Lipase

2-1. Gene and amino acid sequence of SKE3 strain showing esterase activity, and phytotoxicity

A restriction map of the plasmid in which the Escherichia active clones were recombined was constructed from the SKE3 strain exhibiting the selectase activity in Example 1, and the nucleotide sequence of the plasmid was analyzed by SolGent (Daejeon, Korea). The nucleotide sequence of the conserved region of the cloned gene was confirmed by using BlastP of NCBI (http://www.ncbi.nlm.nih.gov), CBS (http://www.cbs.dtu.dk/services / SignalP /) SignalP 3.0 was used to predict the signal peptide.

On the other hand, the phylogenetic tree of the enzyme was prepared using DNA / MAN (Lynnon Biosoft, version 4.11, Quebec, Canada). The molecular weight of the coded protein and the isoelectric point (pI) of the encoded protein were analyzed using ExPASy (http://www.expasy.ch/tools/protparam.html) and the active site of the coded protein (active site) were analyzed using the pfam database. The results are shown in Fig. 1 and Fig.

As a result, a DNA fragment of about 3.3 kb in size was inserted into the SKE3 recombinant plasmid, and ORF (open reading frames) encoding an enzyme exhibiting very similar activity to the esterase in the inserted DNA fragment contained a 900 bp polynucleotide ), Respectively. The present inventor named the lipolytic enzyme showing activity very similar to the esterase as Est3K.

Although not shown in the figure, Est3K encodes 299 amino acids (SEQ ID NO: 2), no signal peptide was identified, and the Esterase of Pseudomonas sp. Ag1 (GenBank accession number WP_008438797) and Pseudomonas fluorescens It has 99% homology with the Esterase / Lipase (WP_023659021) of Pseudomonas fluorescens , has 95% homology with Pseudomonas species PAMC 26793 Esterase (WP_026078148), 89% with Esterase (WP_010175806) of Pseudomonas species PAMC 25886, Homology and 76% homology with the esterase of Pseudomonas syringae (WP_027901927).

As shown in FIG. 1, Est3K is an HGGG (oxymoron hole) motif (74th to 78th amino acid), GXSXG motif (144th to 148th amino acid), DPL motif 244 And the HGF motif (270th to 272nd amino acid) region, and the molecular weight and theoretical isoelectric point of Est3K are estimated to be 32,354 Da and 8.82, respectively.

In addition, as shown in FIG. 3, Est3K is confirmed to belong to Family IV through the creation of phonemes.

2-2. Lipase activity gene and amino acid sequence of SKEL5 strain exhibiting esterase activity and lipase activity, and phylogenetic tree preparation

A restriction map of the plasmid in which the lipase active clones were recombined was constructed from the SKEL5 strain showing the Esterase activity and the lipase activity selected in Example 1, and the nucleotide sequence of the plasmid was assigned to SolGent (Daejeon, Korea) , The sequence of the gene having lipase activity and the signal peptide, the amino acid sequence of the lipase-activating enzyme, the molecular weight, the theoretical isoelectric point (pI) and the phylogenetic tree were prepared in the same manner as in 2-1 above. The results are shown in Fig. 2 and Fig.

As a result, it was confirmed that the ORF (open reading frames: lip 3K) coding for lipase in the SKEL5 recombinant plasmid was composed of a total of 1,951 bp polynucleotide (SEQ ID NO: 3). The present inventor named the lipolytic enzyme showing lipase activity as Lip3K.

Although not shown in the figure, Lip3K encodes 616 amino acids (SEQ ID NO: 4), no signal peptide was identified, and the estimated calcium-phosphorylation of Pseudomonas species UB48 (AAG09700) lipase, Pseudomonas species GM30 (WP_007967001) And 92% homology with the lipase of Pseudomonas fluorescens B52 (AAT48728) and Pseudomonas species CR-611 (AFP20145), which has 93% homology with the binding protein and the lipase of Pseudomonas sp. MIS38 (BAA84997) .

As shown in Fig. 2, Lip3K has 5 residues sequence motif VTLVG (598th to 602nd), 9 residue sequence motif GGxGxDxux (372nd to 416th, 492nd to 545th) in the conserved region of lipase belonging to Family 1.3 (54th to 556th) and GxSxG (204th to 208th) regions, and the molecular weight and theoretical isoelectric point of Lip3K are estimated to be 64,408 Da and 4.53, respectively.

In addition, as shown in FIG. 3, Lip3K was confirmed to belong to Family I.3 through generation of phylogenetic trees.

Example 3: Est3K or Lip3K purification

The SKE3 strain having the esterase activity and the SKEL5 strain having the esterase activity and the lipase activity selected in Example 1 were suspended in an LB liquid medium (10 g / L Bacto trypton, 5 g / L ampicillin containing 100 g / ml ampicillin, L yeast extract, 10 g / L NaCl) and cultured at 37 ° C in a shaking incubator at a speed of 200 rpm for 12 hours. Then, Jeong et al . (2012, Applied Biochemistry and Biotechnology, 166: 1328-1339. , The coenzyme solution was recovered. (5 ml, Bio-Rad, California, USA), CHT-II column (5 ml, Bio-Rad, California, USA) and t-ButylHIC column (5 ml, Bio- Rad, California, USA) Bio-Rad, California, USA) to isolate Est3K. The coenzyme solution recovered from the SKEL5 strain was passed through a High-Q column (5 ml, Bio-Rad, California, USA) to isolate Lip3K. The active fractions passed through the column were immersed in a heated water bath for 1 minute and then separated on a 11.5% polyacrylamide gel. Then, the gel was washed twice in isopropanol, twice in 50 mM Tris-HCl (pH 8.0), and then loaded on an agar strip containing 1% glyceryl trioctanoate at 50 ° C. for 1 hour Lip3K was confirmed. Lip3K identified in the gel was orchard, chopped, and eluted at 4 ° C for one day to analyze the amount of protein. The results are shown in Fig.

As shown in Fig. 4, a single band was confirmed at a size of about 34 kDa in Est3K, and the amount thereof was confirmed to be 8.4 U / mg. On the other hand, the single band of Lip3K was confirmed at about 64 kDa and its amount was found to be 10.3 U / mg.

Example 4: Substrate specificity of Est3K or Lip3K

Activity of the above-described one or Est3K Lip3K purified in Example 3, p - nitrophenyl ester from (p -nitrophenyl esters; p-nitrophenyl caproate from Tokyo Chemical Industry Co., Tokyo, Japan; Sigma, St. Louis, MO, USA) separate p - the amount of the nitrophenol (p -nitrophenol) was confirmed by measurement. Specifically, the esterase or lipase of the Est3K Lip3K 1 mM concentrations each of p - nitrophenyl ester (p -nitrophenyl esters), p - nitrophenyl acetate (p -nitrophenyl acetate; C2), p - nitrophenyl butyrate (p -nitrophenyl butyrate ; C4), p - nitrophenyl caproate (p -nitrophenyl caproate; C6), p - nitrophenyl octanoate (p -nitrophenyl octanoate: C8), p - nitrophenyl caprate (p -nitrophenyl caprate; C10), p - nitrophenyl laurate (p -nitrophenyl laurate; C12), p - nitrophenyl pre stearyl acrylate (p -nitrophenyl myristate; C14), and p - nitrophenyl palmitate; 50 contain (p -nitrophenyl palmitate C16) The reaction was carried out at 25 ° C for 1 minute and then the activity of Est3K or Lip3K was measured at 450 nm using a spectrophotometer (Mecasys, Model OPTIZEN, KOREA). The activity of Est3K or Lip3K was expressed as a percentage using the absorbance coefficient of p - nitrophenol. One unit of Est3K or Lip3K was defined as the amount in which 1 μmol of p - nitrophenol was measured for 1 minute. Further, the extinction coefficient of p -nitrophenol was used, and the results are shown in Fig. 5 and Table 1. Fig.

Enzyme activity (A / min) Est3K Lip3K Est3K + Lip3K p NP-butyrate (C4) 0.522 (1.00) 0.301 (1.00) 0.711 (1.00) p NP-octanoate (C8) 0.364 (0.70) 0.465 (1.54) 0.703 (0.99)

As shown in FIG. 5, Est3K are short-chain fatty acids in the p - and decomposing nitrophenyl (p NP) ester hydrolysis, Lip3K is a medium chain fatty acid p - exhibited a hydrolytic activity of a dinitrophenyl (p NP) ester.

Specifically, Est3K the p - nitrophenyl butyrate (p NP-butyrate; C4) , p - nitrophenyl caproate (p NP-caproate; C6) , p - nitrophenyl octanoate (p NP-octanoate; C8) and The activity of decomposing p -nitrophenylacetate ( p NP-acetate; C2) was 100%, 78.0%, 70.1% and 60.7%, respectively, and Lip3K was found to be p -nitrophenyl octanoate ( p NP-octanoate; C8), p - nitrophenyl caprate (p NP-caprate; C10) , p - nitrophenyl acetate (p NP-acetate; C2) , p - nitrophenyl butyrate (pNP-butyrate; C4) and p - nitrophenyl caproate ( P NP-caproate; C6) were 100%, 78.9%, 69.3%, 67.0%, and 60.4%, respectively.

Meanwhile, as shown in Table 1, the activity ratio of Est3K to C4 and C8 hydrolysis was 1: 0.7, and the activity ratio of Lip3K to C4 and C8 hydrolysis was 1: 1.5. In addition, the activity ratio of the mixture of the same amounts of Est3K and Lip3K hydrolyzed into C4 and C8 was 1: 0.99.

These results indicate that Est3K and Lip3K of the present invention have excellent lipid hydrolysis efficiency and that the two enzymes do not show synergistic effects with each other.

Example 5 Temperature, Thermal Stability and pH Selection Experiment of Est3K or Lip3K Optimal Activity

5-1. Optimal temperature selection experiment of Est3K or Lip3K

Est3K or Lip3K purified in Example 3 was treated with p -nitrophenyl p -nitrophenyl butyrate ( p NP-butyrate; C4) and p -nitrophenyl octanoate ( p NP-octanoate; (50 mM Tris-HCl, pH 8.0) and the relative activity was measured at 10 ° C intervals for a temperature ranging from 30 to 70 ° C. The results are shown in Fig.

As shown in Fig. 6, Est3K showed the optimum activity at 50 캜 and 85% activity at 40 캜.

On the other hand, Lip3K showed the optimum activity at 50 ° C and less than 50% at other temperatures.

5-2. Temperature stability experiment of Est3K or Lip3K

Est3K or Lip3K purified in Example 3 was treated with p -nitrophenyl p -nitrophenyl butyrate ( p NP-butyrate; C4) and p -nitrophenyl octanoate ( p NP-octanoate; (50 mM Tris-HCl, pH 8.0) and stored at a temperature ranging from 30 to 70 ° C for 1 hour, and then the activity thereof was measured. The results are shown in Fig.

As shown in Fig. 7, the activity of Est3K was about 100% even when stored at 30 DEG C for 1 hour. However, when stored at 40 ° C for 30 minutes, the activity decreased to 62%, and when stored at 50 ° C for 30 minutes, the activity was almost not observed.

The activity of Lip3K was about 100% even when stored at 30 ° C for 1 hour. However, when stored at 40 ° C or 50 ° C for 30 minutes, the activity was reduced to 64% and 50%, respectively, and it was confirmed that the activity was almost not exhibited when stored at 60 ° C for 30 minutes.

5-3. Optimal pH selection experiment of Est3K or Lip3K

Est3K or Lip3K purified in Example 3 was treated with p -nitrophenyl p -nitrophenyl butyrate ( p NP-butyrate; C4) and p -nitrophenyl octanoate ( p NP-octanoate; The activity was measured by reacting 50 mM Tris-HCl (pH 8.0) with boric acid / citric acid / trisodium orthophosphate. The results are shown in Fig.

As shown in Fig. 8, the optimum pH of Est3K and Lip3K was confirmed at pH 9.0.

Example 6: Effect of metal ion or organic solvent on the activity of Est3K or Lip3K

6-1. Effect of monovalent or divalent metal ions on the activity of Est3K or Lip3K

Of each optimum substrate Example 3 or one Est3K Lip3K purified in ^{5 mM K +, Na +,} Ca 2+, Mg 2+, Mn 2+, Zn 2+, or was previously reacted with Cu ²⁺ p - nitrophenyl p - nitrophenyl butyrate (p NP-butyrate; C4) and p - nitrophenyl octanoate (p NP-octanoate; C8) measuring the 50 mM Tris-HCl (pH 8.0 ) into the active and stored in containing the Respectively. As a control, 1% isopropanol was used instead of metal ions. The results are shown in Table 2.

Enzyme activity (%) Est3K Lip3K Control (+ 1% isopropanol) 100.0 100.0 Metal ions (5 mM) K ⁺ 93.9 ± 4.5 89.9 ± 2.2 Na ⁺ 92.2 ± 3.8 103.0 + - 2.8 Ca ²⁺ 91.1 ± 2.0 190.9 ± 7.1 Mg ²⁺ 88.4 ± 2.5 122.2 ± 1.3 Mn ^{2 +} 86.7 ± 4.2 83.8 ± 1.6 Zn ²⁺ 85.0 ± 2.3 49.5 ± 5.3 Cu ²⁺ 59.3 ± 5.7 16.2 ± 3.4

As shown in Table 2, most monovalent and divalent metal ions did not significantly affect the activities of Est3K and Lip3K, but 5 mM Cu ^{2 +} inhibited the activities of Est3K and Lip3K by 40.7% and 83.8%, respectively And 5 mM Zn ^{2 +} inhibited the activity of Lip3K by 50.5%. On the other hand, 5 mM Ca ²⁺ increased the activity of Lip3K by 109.9%.

6-2. Effect of Ca ²⁺ ion on the activity of Lip3K

Example 3 After a purification Lip3K was previously reacted with 5 mM Ca ²⁺ from the substrate p - nitrophenyl octanoate (p -nitrophenyl octanoate: C8) of 50 mM Tris-HCl into a (pH 8.0) containing the And stored at a temperature in the range of 30 to 60 ° C for 1 hour to measure its activity. The results are shown in Fig.

As shown in FIG. 9, when Lip3K was stored at 60 DEG C for 15 minutes in the presence of Ca2 ⁺ , the activity of Lip3K decreased by 33%. On the other hand, as shown in FIG. 7, when Lip3K was stored at 60 DEG C for 15 minutes without Ca2 ⁺ , the activity of Lip3K decreased by 82%.

In other words, Ca ²⁺ increased the activity of Lip3K at high temperature.

6-3. Effect of organic solvent on the activity of Est3K or Lip3K

After the reaction of Est3K or Lip3K purified in Example 3 with 5% isopropanol, 30% isopropanol, 5% methanol, 30% methanol, 5% acetonitrile or 30% acetonitrile, p - nitrophenyl p -nitrophenyl butyrate (p NP-butyrate; C4) and p-nitrophenyl octanoate; to (p NP-octanoate C8) into a 50 mM Tris-HCl (pH 8.0 ) containing the storage was measured for its activity. The results are shown in Table 3.

Enzyme activity (%) Est3K Lip3K Control (+ 1% isopropanol) 100.0 100.0 Organic solvent Isopropanol (5%) 99.2 ± 2.5 109.1 ± 2.8 Isopropanol (30%) 87.5 ± 4.4 114.1 ± 2.0 Methanol (5%) 93.8 ± 3.5 101.0 ± 2.7 Methanol (30%) 86.9 ± 2.2 166.7 ± 4.8 Acetonitrile (5%) 49.6 ± 5.6 30.3 ± 3.0 Acetonitrile (30%) 47.7 ± 5.1 37.4 ± 4.6

As shown in Table 3 above, Est3K exhibited activity of 85% or more in the presence of 5% isopropanol or methanol, 30% of isopropanol or methanol, but exhibited activity of 50% or less in the presence of 5% acetonitrile and 30% acetonitrile Respectively.

On the other hand, the activity of Lip3K was improved in the presence of 5% of isopropanol or methanol, 30% of isopropanol or methanol, and the activity was improved by 14.1% and 66.7% in the presence of 30% isopropanol and 30% methanol, respectively. However, Lip3K also showed activity of 40% or less in the presence of 5% acetonitrile and 30% acetonitrile.

From the above results, it can be seen that Est3K or Lip3K is a novel lipolytic enzyme showing low homology and characteristics with conventional lipolytic enzymes derived from microorganisms or metagenomes of forest soils. The nucleotide sequence homology and characteristics of the conventional lipolytic enzyme and the Est3K or Lip3K of the present invention are shown in Table 4 below.

<110> Sunchon University Industry-Academic Cooperation Foundation &Lt; 120 > {Novel lipolytic enzyme from metagenomic library and process for          preparing the same <130> PA-15-0201 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 900 <212> DNA <213> Artificial Sequence <220> <223> ORF sequence of est3K gene <400> 1 atgcagcgct ttaaccagaa gctcgcctgg atgccgcgtt ttcgcatccg caaccgcgtg 60 acgccccggg tgatccaggc gctgttgcgc tccagccaga tggtggccgg caacaagttg 120 ctcaagcatg gcctgcaggc tgaaagccgg cgggtgggct cggtgccggt gcgcatcatc 180 cggccgaagg gcaaggccaa aggcgtggtg ctggatatcc atggcggcgg ctgggtgatc 240 ggcaatgcgc agatgaatga cgacctcaac gtggccatgg tgaatgcgtg cgaggtggcg 300 gtggtgtcgg tggattaccg gctggcggtg aatacgccgg tcgaagggat tctggaagac 360 tgcctggcca cggcgcgctg gttgctggcg gactgtgagg agttcgccgg cctgccggtg 420 atcgttgtcg gcgagtctgc cggtgggcat ctggcggcgg ccacgctgct ggcgctgaag 480 caatcgcccg aattgctggc gcgggtgagc ggggcggtgc tgtattacgg cgtctacgat 540 ttgaccggta cgccgagcgt gcgcacggca ggacgcgaaa cgctgctgct ggacgggccg 600 ggcatggtgg aggcgctgcg tttgctgacg ccgggcctga gtgacgagca gcgccggcag 660 ccgccgttgt caccgttgta tggcgaccta gccgggctgc cgccagcgct gatgtttgtg 720 ggggagctgg acccgttgct ggacgacacg ctgcagatgg ccgagcgatg ggcggggtcc 780 gggtgtttt tgttaccgca ggcggctcat gggtttattc attttccgac ggcgatgtcg 840 ggccgcgtgc tggcttacag tcgcgagtgg ataacgggtc gtttacgctc ggttggttga 900                                                                          900 <210> 2 <211> 299 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Est3K <400> 2 Met Gln Arg Phe Asn Gln Lys Leu Ala Trp Met Pro Arg Phe Arg Ile   1 5 10 15 Arg Asn Arg Val Thr Pro Arg Val Ile Gln Ala Leu Leu Arg Ser Ser              20 25 30 Gln Met Val Ala Gly Asn Lys Leu Leu Lys His Gly Leu Gln Ala Glu          35 40 45 Ser Arg Arg Val Gly Ser Val Pro Val Arg Ile Ile Arg Pro Lys Gly      50 55 60 Lys Ala Lys Gly Val Val Leu Asp Ile His Gly Gly Gly Trp Val Ile  65 70 75 80 Gly Asn Ala Gln Met Asn Asp Asp Leu Asn Val Ala Met Val Asn Ala                  85 90 95 Cys Glu Val Ala Val Val Ser Val Asp Tyr Arg Leu Ala Val Asn Thr             100 105 110 Pro Val Glu Gly Ile Leu Glu Asp Cys Leu Ala Thr Ala Arg Trp Leu         115 120 125 Leu Ala Asp Cys Glu Glu Phe Ala Gly Leu Pro Val Ile Val Val Gly     130 135 140 Glu Ser Ala Gly Gly His Leu Ala Ala Ala Thr Leu Leu Ala Leu Lys 145 150 155 160 Gln Ser Pro Glu Leu Leu Ala Arg Val Ser Gly Ala Val Leu Tyr Tyr                 165 170 175 Gly Val Tyr Asp Leu Thr Gly Thr Pro Ser Val Arg Thr Ala Gly Arg             180 185 190 Glu Thr Leu Leu Leu Asp Gly Pro Gly Met Val Glu Ala Leu Arg Leu         195 200 205 Leu Thr Pro Gly Leu Ser Asp Glu Gln Arg Arg Gln Pro Pro Leu Ser     210 215 220 Pro Leu Tyr Gly Asp Leu Ala Gly Leu Pro Pro Ala Leu Met Phe Val 225 230 235 240 Gly Glu Leu Asp Pro Leu Leu Asp Asp Thr Leu Gln Met Ala Glu Arg                 245 250 255 Trp Ala Gly Ser Glu Val Phe Leu Leu Pro Gln Ala Ala His Gly Phe             260 265 270 Ile His Phe Pro Thr Ala Met Ser Gly Arg Val Leu Ala Tyr Ser Arg         275 280 285 Glu Trp Ile Thr Gly Arg Leu Arg Ser Val Gly     290 295 <210> 3 <211> 1851 <212> DNA <213> Artificial Sequence <220> <223> ORF sequence of lip3K gene <400> 3 atgggtgtgt acgactacaa gaacttcagc tcagcggatt ccaaggcgtt gttcactgat 60 gccatggcga tcacgctgta ctcctatcac aacctcgaca acggtttcgc tgtcgggtat 120 cagcacaacg gcttcggcct cggcctgccg gccacgctgg tttcggcgct gatcggcggc 180 acggattcgc aaggcgtgat tccggactcc gtggaccccg actcggaaaa actcgctctg 240 gacgccgtga aaaaggctgg ctggacgccc atcaccgcct cgcaactggg ctatgacggc 300 aagaccgacg ctcgcggaac cttcttcggc gaaaaggccg gctacaccag cgcgcaagtg 360 gaaatcctcg gcaagtacga cgcccagggt catctcacgg aaatcggcat cgcctttcgc 420 ggcaccagcg gcccgcggga aatcctgatc ggcgactcca tcgccgacgc catcaatgac 480 ctgctcgccg cgttcggccc caaggattac gccaagaact acgttggtga agccttcggc 540 aacctgctga acgatgtggt ggccttcgcc aaggccaacg gtctcaccgg caaggacgtg 600 ctggtcagtg gtcacagcct cggcggcctg gcggtcaaca gcatggcgga cttgagcggc 660 ggcaaatggg gcgggttctt ccaggactcc aactacatcg cctacgcctc accgacccaa 720 agcagcaccg acaaagtgct caacgtcggc tacgagaacg acccggtctt ccgcgcgctc 780 gacggctcga ccttcaccgg cgcctcgctt ggcgtacacg acgcgtcgaa agtgtcggcg 840 accgacaaca tcgtcagctt caacgaccac tacgcttcga acgtgtggaa cgtgctgcca 900 ttctcgatcg tcaacgtccc gacctggatc tcgcacctgc cgacagccta cggcgacggc 960 atgaccggg tgatcgagtc gaagttctac gatctcacca gccgtgactc gacgatcatc 1020 gtcgccaacc tgtcggaccc ggcgcgggcc aacacctggg tgcaggacct caaccgcaac 1080 gccgaaaccc acaagggcag caccttcatc atcggcagcg acggcaacga cctgatccag 1140 ggcggcagcg gcaatgacta ccttgaagga cgggccggca acgacacgtt ccgtgacagc 1200 ggcggctaca acgtcattct tggcggtgcc ggcaacaaca ccctcgactt gcagcagtcg 1260 gtgaacagt tcgacttcgc caacgacggc gccggcaacc tgtacatccg cgatgccaac 1320 ggcgggatca gcatcacccg cgacatcggc agcatcgtca ccaaggagcc gggtttcctc 1380 tggggcctgt tcaaggacga cgtgacccac agcgtcacgg cggatggcct gaaggtcggc 1440 aacaacgtca cccagtacga gtcgagcgtg aagggcacca gtggcgtcga caccctcaag 1500 gcccacgcca gcggtgactg gctgttcggc ctggacggca acgatcacct gatcggtggc 1560 gtgggcaacg acgtgtttgt cggcggcgcc ggcaatgacc tgatggagtc gggtggcggg 1620 gcggatacgt tcctgttcaa cggcgcgttc ggccaggatc gggtggtggg attcaccggc 1680 aacgacaaac tggtgtttct cggcgtgcag ggcgtgttgc cgggcgatga cttccgcgcc 1740 catgccacct cggtggggca ggatacggtg ctgaagttcg gcggtgattc cgtgacgctg 1800 gtcggggttt cgctgagcag tctcagtgcc gatggcatcg ttatcgcctg a 1851 <210> 4 <211> 616 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of Lip3K <400> 4 Met Gly Val Tyr Asp Tyr Lys Asn Phe Ser Ser Ala Asp Ser Lys Ala   1 5 10 15 Leu Phe Thr Asp Ala Met Ala Ile Thr Leu Tyr Ser Tyr His Asn Leu              20 25 30 Asp Asn Gly Phe Ala Val Gly Tyr Gln His Asn Gly Phe Gly Leu Gly          35 40 45 Leu Pro Ala Thr Leu Val Ser Ala Leu Ile Gly Gly Thr Asp Ser Gln      50 55 60 Gly Val Ile Pro Asp Ser Val Asp Pro Asp Ser Glu Lys Leu Ala Leu  65 70 75 80 Asp Ala Val Lys Lys Ala Gly Trp Thr Pro Ile Thr Ala Ser Gln Leu                  85 90 95 Gly Tyr Asp Gly Lys Thr Asp Ala Arg Gly Thr Phe Phe Gly Glu Lys             100 105 110 Ala Gly Tyr Thr Ser Ala Gln Val Glu Ile Leu Gly Lys Tyr Asp Ala         115 120 125 Gln Gly His Leu Thr Glu Ile Gly Ile Ala Phe Arg Gly Thr Ser Gly     130 135 140 Pro Arg Glu Ile Leu Ile Gly Asp Ser Ile Ala Asp Ala Ile Asn Asp 145 150 155 160 Leu Leu Ala Ala Phe Gly Pro Lys Asp Tyr Ala Lys Asn Tyr Val Gly                 165 170 175 Glu Ala Phe Gly Asn Leu Leu Asn Asp Val Ala Phe Ala Lys Ala             180 185 190 Asn Gly Leu Thr Gly Lys Asp Val Leu Val Ser Gly His Ser Leu Gly         195 200 205 Gly Leu Ala Val Asn Ser Met Ala Asp Leu Ser Gly Gly Lys Trp Gly     210 215 220 Gly Phe Phe Gln Asp Ser Asn Tyr Ile Ala Tyr Ala Ser Pro Thr Gln 225 230 235 240 Ser Ser Thr Asp Lys Val Leu Asn Val Gly Tyr Glu Asn Asp Pro Val                 245 250 255 Phe Arg Ala Leu Asp Gly Ser Thr Phe Thr Gly Ala Ser Leu Gly Val             260 265 270 His Asp Ala Ser Lys Val Ser Ala Thr Asp Asn Ile Val Ser Phe Asn         275 280 285 Asp His Tyr Ala Ser Asn Val Trp Asn Val Leu Pro Phe Ser Ile Val     290 295 300 Asn Val Pro Thr Trp Ile Ser His Leu Pro Thr Ala Tyr Gly Asp Gly 305 310 315 320 Met Asn Arg Val Ile Glu Ser Lys Phe Tyr Asp Leu Thr Ser Arg Asp                 325 330 335 Ser Thr Ile Val Ala Asn Leu Ser Asp Pro Ala Arg Ala Asn Thr             340 345 350 Trp Val Gln Asp Leu Asn Arg Asn Ala Glu Thr His Lys Gly Ser Thr         355 360 365 Phe Ile Ile Gly Ser Asp Gly Asn Asp Leu Ile Gln Gly Gly Ser Gly     370 375 380 Asn Asp Tyr Leu Glu Gly Arg Ala Gly Asn Asp Thr Phe Arg Asp Ser 385 390 395 400 Gly Gly Tyr Asn Val Ile Leu Gly Gly Ala Gly Asn Asn Thr Leu Asp                 405 410 415 Leu Gln Gln Ser Val Asn Lys Phe Asp Phe Ala Asn Asp Gly Ala Gly             420 425 430 Asn Leu Tyr Ile Arg Asp Ala Asn Gly Gly Ile Ser Ile Thr Arg Asp         435 440 445 Ile Gly Ser Ile Val Thr Lys Glu Pro Gly Phe Leu Trp Gly Leu Phe     450 455 460 Lys Asp Asp Val Thr His Ser Val Thr Ala Asp Gly Leu Lys Val Gly 465 470 475 480 Asn Asn Val Thr Gln Tyr Glu Ser Ser Val Lys Gly Thr Ser Gly Val                 485 490 495 Asp Thr Leu Lys Ala His Ala Ser Gly Asp Trp Leu Phe Gly Leu Asp             500 505 510 Gly Asn His Leu Ile Gly Gly Val Gly Asn Asp Val Phe Val Gly         515 520 525 Gly Ala Gly Asn Asp Leu Met Glu Ser Gly Gly Gly Ala Asp Thr Phe     530 535 540 Leu Phe Asn Gly Ala Phe Gly Gln Asp Arg Val Val Gly Phe Thr Gly 545 550 555 560 Asn Asp Lys Leu Val Phe Leu Gly Val Gln Gly Val Leu Pro Gly Asp                 565 570 575 Asp Phe Arg Ala His Ala Thr Ser Val Gly Gln Asp Thr Val Leu Lys             580 585 590 Phe Gly Gly Asp Ser Val Thr Leu Val Gly Val Ser Leu Ser Ser Leu         595 600 605 Ser Ala Asp Gly Ile Val Ile Ala     610 615

Claims (10)

2, having a molecular weight of 34 kDa, an optimal activity temperature and a pH of 50 DEG C and a pH of 9.0, respectively, and having an alkaline and heat-stable enzyme activity stable at pH 8.0 to 10.0 and 40 to 55 DEG C, A novel esterase (Est3K) characterized by selectively hydrolyzing p -nitrophenylbutyrate (C4).
delete A polynucleotide encoding the esterase of claim 1 (Est3K).
A recombinant vector comprising the polynucleotide of claim 3.
A transformant transformed by introducing the recombinant vector of claim 4.
delete delete delete a) culturing the transformant of claim 5 or a cell having the polynucleotide of claim 3; b) inducing expression of an esterase (Est3K) in said transformant or cells; c) eluting the Escherase (Est3K) from the supernatant obtained by pulverizing and then precipitating the transformant or cells expressing the Escherase (Est3K); And d) purifying the eluted Esterase (Est3K).
delete

Images