KR102227399B1 - Novel thermostable phospholipase A2 and process for preparing phospholipase A2 - Google Patents

Abstract

The present invention relates to novel phospholipase A2 (PLA2) having heat resistance. More specifically, the present invention relates to PLA2 which is a novel enzyme having heat resistance, a polynucleotide encoding the enzyme, an expression vector comprising the polynucleotide, a transformant capable of producing the enzyme by introducing the expression vector, and a method for producing the enzyme using the transformant. The PLA2 of the present invention has heat resistance, and thus can be widely used for production, development, and utilization of the PLA2.

Description

New heat-resistant phospholipase A2 and phospholipase A2 production method {Novel thermostable phospholipase A2 and process for preparing phospholipase A2}

The present invention relates to a novel phospholipase (Phospholipase A2; PLA2) enzyme having heat resistance, and more specifically, the present invention, a novel PLA2 enzyme having heat resistance, a polynucleotide encoding the enzyme, and the polynucleotide. It relates to an expression vector, a transformant capable of producing the enzyme by introducing the expression vector, and a method of producing the enzyme using the transformant.

Phospholipase is an enzyme that hydrolyzes phospholipids and is also called recitinase. Phospholipase is classified into A1, A2, B, C, D depending on the position where it degrades phospholipids, and the phospholipase A2 component of the phospholipase is a fatty acid that binds to the BDP of glycerin of phospholipid, a lipid constituting the cell membrane. It is known to cause cell tissue destruction, hemolysis, and catalytic action because it is released and made into lyzophosphatide. Various pharmaceutical compositions can be developed using this action of phospholipase A2, and Korean Patent Publication No. 10-2015-0049438 proposes a composition capable of preventing and treating viral diseases such as influenza and vesicular stomatitis. have.

On the other hand, the most important thing in a biological process in which an enzyme is used as a biological catalyst to produce useful substances is the stability of the enzyme. In particular, since it is known that the biocatalytic reaction performed at a high temperature has a relatively high production yield compared to the enzyme reaction performed at a low temperature, the thermal stability of the enzyme in a biological process is a very important problem. In addition, compared to reactions at room temperature or low temperature, high-temperature enzymatic reactions have advantages such as higher substrate solubility of enzymes, reduced microbial contamination, and decreased viscosity of the solution. The development is very useful for bioprocesses and industries that apply them. In addition, it is known that heat-resistant enzymes with increased stability against heat can improve structural stability at room temperature, stability against organic solvents, stability against extreme hydrogen ion concentration, and stability against protein denaturants.

Therefore, in order to actively utilize phospholipase A2 in a bioprocess, the need to improve the thermal stability of phospholipase A2 has emerged.

Under this background, the present inventors, as a result of continuing efforts to develop phospholipase A2 with improved thermal stability, confirmed that the phospholipase A2 derived from Sciscionella marina and Marinoactinospora thermotolerans has heat resistance, thereby completing the present invention. .

One object of the present invention is to provide a heat-resistant phospholipase A2 enzyme comprising the amino acid sequence of SEQ ID NO: 1 or 2.

Another object of the present invention is to provide a polynucleotide encoding the phospholipase A2.

Another object of the present invention is to provide a vector for expression of phospholipase A2 comprising the polynucleotide.

Another object of the present invention is to provide a transformant capable of producing the phospholipase A2 by introducing the vector.

Another object of the present invention is to provide a method for producing the phospholipase A2 using the transformant.

One embodiment of the present invention for achieving the above object provides a heat-resistant phospholipase A2 enzyme comprising the amino acid sequence of SEQ ID NO: 1 or 2.

In the present invention, the term "phospholipase" refers to phospholipase, which is an enzyme that hydrolyzes phospholipids and is also referred to as recitinase. Phospholipase is classified into A1, A2, B, C, D depending on the position where it degrades phospholipids. Among the phospholipases, the "phospholipase A2 (PLA2)" of the present invention is It is known to cause the destruction of cellular tissues, hemolysis and catalysis, etc., because glycerin releases BDP-binding fatty acids to make lyzophosphatide.

The PLA2 may be derived from Sciscionella marina containing an amino acid sequence (SEQ ID NO: 1), and from Marinoactinospora thermotolerans containing an amino acid sequence (SEQ ID NO: 2). It may be of origin. Specifically, the PLA2 may be made of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In addition, the PLA2 enzyme comprising the amino acid sequence of SEQ ID NO: 1 may have an optimal bioconversion reaction activity at pH 7.5 and temperature 50 °C, and the PLA2 enzyme comprising the amino acid of SEQ ID NO: 2 is at pH 7.5 and temperature 50 °C. It may have an optimal biotransformation reaction activity.

In the present invention, PLA2 having heat resistance is characterized by increased heat resistance compared to PLA2 derived from Streptomyces violaceoruber. The PLA2 is 50? It may be an enzyme that has heat resistance even at high temperature culture at 60°C. Specifically, in an embodiment of the present invention, PLA2 derived from Streptomyces violaceoruber, Sciscionella marina, and Marinoactinospora thermotolerans was cultured at various temperatures to analyze the optimum activity temperature of the biotransformation reaction, confirming that the optimum temperature of the biotransformation reaction activity was relatively high. (Figs. 11, 13 and 14), the PLA2 transformants derived from each were cultured in a shaking incubator at 50°C and 60°C, and compared the activity of the produced PLA2, it was confirmed that the new PLA2 enzymes were relatively high in heat resistance. (Figs. 12 and 15).

Another embodiment of the present invention for achieving the above object is a polynucleotide encoding the PLA2 enzyme, a PLA2 enzyme expression vector comprising the polynucleotide, a transformant into which the expression vector is introduced, and the transformant. It provides a method of preparing the PLA2.

The polynucleotide provided by the present invention encodes the PLA2 enzyme provided by the present invention, and specifically, may include SEQ ID NO: 3 to SEQ ID NO: 4. The polynucleotide may be modified by substitution, deletion, insertion, or a combination of one or more bases. When preparing by chemically synthesizing a nucleotide sequence, a synthetic method well known in the art, for example, a method described in Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988, can be used. , Tryester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis methods on a solid support.

The term "expression vector" of the present invention refers to a recombinant vector capable of expressing a peptide of interest in a host cell of interest, and refers to a gene construct comprising essential regulatory elements operably linked so that a gene insert is expressed. The expression vector includes expression control elements such as a start codon, a stop codon, a promoter, and an operator, and the start and stop codons are generally considered to be part of the nucleotide sequence encoding the polypeptide, and when the gene construct is administered, the individual It must exhibit an action in and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible.

The term "operably linked" of the present invention means a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked to perform a general function. . For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect the expression of the coding sequence. The operative linkage with the expression vector may be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage may use enzymes generally known in the art.

In addition, the expression vector may include a signal sequence for the release of the fusion polypeptide in order to facilitate the separation of the protein from the cell culture medium. Specific initiation signals may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG initiation codon and adjacent sequences. In some cases, exogenous translational control signals should be provided, which may include the ATG initiation codon. These exogenous translational control signals and initiation codons can be of a variety of natural and synthetic sources. Expression efficiency can be increased by introduction of appropriate transcriptional or translation enhancing factors.

In addition, the expression vector may additionally include a protein tag that can be removed using an endopeptase optionally in order to facilitate detection of the PLA2 enzyme.

The term "tag" of the present invention means a molecule that exhibits quantifiable activity or properties, and a polypeptide fluorescent substance such as a chemical fluorescent substance such as fluorescein, a fluorescent protein (GFP), or a related protein It may be a fluorescent molecule including; It may be an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, or a strapavidin tag. In particular, when using an epitope tag, preferably a peptide tag consisting of 6 or more amino acid residues, more preferably 8 to 50 amino acid residues may be used.

In the present invention, the expression vector may include a nucleotide sequence encoding the PLA2 enzyme provided in the present invention described above, and the vector used at this time is not particularly limited thereto as long as it can prepare the PLA2 enzyme of the present invention. However, preferably, it may be plasmid DNA, phage DNA, and the like, more preferably commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retro It can be a virus (retrovirus), adenovirus (adenovirus), vaccinia virus (vaccinia virus, etc.), insect virus vector (baculovirus (baculovirus), etc.). Since the expression vector has different expression levels and expressions of proteins depending on the host cell, it is preferable to select and use the most suitable host cell for the purpose.

The transformant provided by the present invention is produced by introducing and transforming the expression vector provided by the present invention into a host, and can be used to prepare the PLA2 enzyme of the present invention by expressing the polynucleotide contained in the expression vector. have. The transformation may be performed by various methods, as long as the PLA2 enzyme of the present invention having heat resistance can be prepared, but is not particularly limited thereto, but a reducing material called dimethyl sulfoxide (DMSO) in the CaCl2 precipitation method and the CaCl2 precipitation method. Hanahan method, electroporation, calcium phosphate precipitation method, protoplasm fusion method, stirring method using silicon carbide fiber, Agrobacteria mediated transformation method, transformation method using PEG, dextran Sulfate, lipofectamine and drying/inhibition mediated transformation methods and the like can be used. In addition, the host used for the production of the transformant is also not particularly limited as long as it can produce the PLA2 enzyme of the present invention, but bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium ; Yeast cells such as Saccharomyces cerevisiae and Schizocaromyces pombe; Fungal cells such as Pichia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, and Bow melanoma cells; Or it could be a plant cell. For example, in the present invention, the E. coli BL21star (DE3) strain was used as a host, but the present invention is not limited thereto.

The transformant can be used in the method of producing the heat-resistant PLA2 enzyme provided by the present invention. Specifically, the method for producing the PLA2 enzyme provided by the present invention includes the steps of: (a) culturing the transformant to obtain a culture; And (b) obtaining the PLA2 enzyme of the present invention from the culture.

The PLA2 enzyme may be produced through secretion outside the cell, and may be activated by a low-purity substrate widely used in the process, but is not limited thereto.

The term "culture" of the present invention means a method of growing microorganisms in an appropriately artificially controlled environmental condition.

In the present invention, the method of culturing the transformant may be performed using a method widely known in the art. Specifically, the culture is not particularly limited as long as it can be prepared by expressing the PLA2 enzyme of the present invention, but it can be continuously cultured in a batch process, an injection batch, or a fed batch or repeated fed batch process. have.

The medium used for cultivation must meet the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc. under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, amino acid, vitamin, and the like. Carbon sources that can be used include sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose, as well as sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose as the main carbon source, soybean oil, sunflower oil, castor oil, coconut oil. Oils and fats such as palmitic acid, stearic acid, fatty acids such as linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials can be used individually or as a mixture. Examples of nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation products, skim soybean cake or its degradation products, etc. I can. These nitrogen sources may be used alone or in combination. The medium may contain first potassium phosphate, second potassium phosphate, and a corresponding sodium-containing salt as personnel. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salt. In addition, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used as the inorganic compound. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

In addition, precursors suitable for the culture medium may be used. The above-described raw materials may be added in a batch, fed-batch, or continuous manner to the culture during the cultivation process, but are not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture.

In addition, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Oxygen or an oxygen-containing gas (eg air) is injected into the culture to maintain an aerobic condition. The temperature of the culture is usually 27°C to 37°C, preferably 30°C to 35°C. The cultivation is continued until the maximum amount of D-type lactic acid is obtained.

For this purpose it is usually achieved in 10 to 100 hours. In general, D-type lactic acid is excreted into the culture medium, but in some cases, it may be contained in the cells.

In addition, the step of recovering the PLA2 enzyme from the culture may be performed by a method known in the art. Specifically, the recovery method is not particularly limited as long as it can recover the prepared PLA2 enzyme of the present invention, but preferably centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, Methods such as fractional dissolution (eg, ammonium sulfate precipitation), chromatography (eg, ion exchange, affinity, hydrophobicity, and size exclusion) can be used.

Since the PLA2 of the present invention has increased heat resistance, it will be widely used in the production, development and utilization of PLA2 enzymes.

1 is a photograph of Western bracketing showing the production amount of SvPLA2.
2 is a graph showing the activity of SvPLA2 based on Pichia pastoris.
3A is a graph showing the results of measuring enzyme titers in cells and in a medium of SvPLA2 according to various E. coli host strains.
3B is a photograph showing the degree of water-soluble expression among the expressed enzymes and the amount of secretion to the medium through SDS-PAGE.
4A is a graph showing the results of measuring SvPLA2 enzyme titer in cells and in a medium according to the presence or absence of codon optimization.
4B is a photograph showing the degree of water-soluble expression among the expressed enzymes and the amount of secretion to the medium through SDS-PAGE.
5 is a graph showing the activity of E. coli-based SvPLA2.
6 is a graph comparing the bioconversion activities of SvPLA2 and SmPLA2 through SDS-PAGE.
7 is a graph showing the bioconversion reaction activity of SvPLA2 by pH condition.
8 is a graph showing the bioconversion reaction activity of SvPLA2 according to temperature conditions.
9 is a graph showing the bioconversion reaction activity of SmPLA2 by pH condition.
10 is a graph showing the bioconversion reaction activity of SmPLA2 according to temperature conditions.
11 is a graph comparing the biotransformation activity of SvPLA2 and SmPLA2 under optimal activity conditions.
12 is a graph comparing the heat resistance of SvPLA2 and SmPLA2.
13 is a graph showing the bioconversion reaction activity of MtPLA2 by pH condition.
14 is a graph showing the bioconversion reaction activity of MtPLA2 according to temperature conditions.
15 is a graph comparing the heat resistance of SvPLA2 and MtPLA2.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1: Production of PLA2 (SvPLA2) enzyme derived from Streptomyces violaceoruber based on Pichia pastoris

Example 1-1: Construction of a recombinant expression vector containing SvPLA2 and a Pichia pastoris transformant

A transformant in which SvPLA2 was produced in Pichia pastoris and secreted extracellularly was produced.

More specifically, by inserting the SvPLA2 enzyme gene into the plasmid vector pPICZαA (Invitrogen, Carlsbad, USA), a pPICZαA-SvPLA2 vector containing the SvPLA2 gene that can be secreted outside the cells of Pichia pastoris was constructed. SvPLA2 was synthesized in Bioneer (Daejeon, Korea) based on the sequence, and the plasmid vector pPICZαA contains the N-terminal α-factor secretion sequence and the C-terminal his tag to secrete the protein produced in the cytoplasm out of the cell. The pPICZαA plasmid vector and SvPLA2 gene having Spe I and Nhe I restriction enzyme sequences were amplified using primers, and the amplified fragment was purified using a PCR purification kit (QIAGEN, Hilden, Germany). The purified PCR product was inserted into a plasmid vector using T4 DNA ligase (New England Biolabs, Ipswich, USA), and in the case of pPICZαA-SvPLA2, 5'-GACTAGTGCTCCTGCAGACAAACC-3' (SEQ ID NO: 5, Spe I) and 3' -CTAGCTAGCTCCAAAAATCTTAACTGCTTGATAATAAGT-5' (SEQ ID NO: 6, Nhe I) primers were prepared and the SvPLA2 gene, 5'-CTAGCTAGCCATCATCATCATCATCATTGAGTTTGT-3' (SEQ ID NO: 7, Nhe I) and 3'- GACTAGTAGCTTCAGCCTCTCTTCTC-5' (SEQ ID NO: 8, SpeⅠ) Primers were prepared to amplify the pPICZαA plasmid vector having Spe I and Nhe I restriction enzyme sequences. The expression vector pPICZαA-SvPLA2 prepared by the above method was used to transform Pichia Pastoris X-33 (Invitrogen) using the Pichia EasyComp Kit (Invitrogen), and the transformed cells were 100 μg/mL of zeocin. (Invitrogen) was grown in YPD medium containing. Positive transformants were identified through PCR and gDNA sequencing.

Example 1-2: Pichia pastoris-based SvPLA2 enzyme production and activity

Fatty acid hydrolysis was performed using the enzyme produced from the transformant.

More specifically, the recombinant Pichia pastoris expressing the SvPLA2 gene was cultured in BMGY medium at 30° C. and 200 rpm for 24 hours, and then the cell pellet obtained by centrifugation at 2000 rpm and room temperature for 1 hour was transferred to BMMY medium. Incubated again. Afterwards, methanol was added at a volume concentration of 1% every 24 hours to induce protein expression. After culturing in BMMY medium for 96 hours, the culture solution was centrifuged to obtain a supernatant, and then a 10-fold concentrated supernatant was obtained using 10kDa Amicon (Millipore, Burlington, USA) and used for fatty acid hydrolysis reaction.

As a result, as shown in FIG. 1, it was confirmed that SvPLA2 was produced in sizes of 14 kDa, 15 kDa, and 18 kDa by western-blot analysis using SDS-PAGE and his tag.

In addition, when the produced SvPLA2 was produced based on the recombinant Pichia pastoris, it was confirmed that soybean-derived lecithin was hydrolyzed into lysolecithin and linoleic acid. More specifically, the fatty acid hydrolysis reaction is 50 mM containing 31.1 g/L L-α-phosphatidylcholine (Sigma-Aldrich, St. Louis, USA), 0.66 g/L calcium chloride aqueous solution, and a concentrated supernatant of 10% volume concentration. Tris-HCl buffer (pH 8.0) was used as a working volume of 10 mL, and reacted at 40° C. and 400 rpm in a stirring mantle (BORK?M, Bayraklı, Turkey). Samples were analyzed by GC/MS.

As a result, as shown in FIG. 2, it was confirmed that 9.6 mM (2.69 g/L) of linoleic acid and lysolecithin were produced as hydrolysis products after 240 minutes of reaction.

Through this, it was confirmed that SvPLA2 based on Pichia pastoris converts L-α-phosphatidylcholine by 32.1%.

Example 2: Transformant selection

The amount of secretion of PLA2 (SvPLA2) derived from Streptomyces violaceoruber using various E. coli hosts was compared to construct a transformant for the extracellular production of PLA2 enzyme.

More specifically, recombinant E. coli expressing the SvPLA2 gene was cultured in Resenberg's medium. 0.2 mM IPTG was added to the culture solution at OD600 = 1.0, and expression was induced at 25° C. and 200 rpm for 24 hours, and the culture solution was centrifuged to obtain a supernatant. Thereafter, a 10-fold concentrated supernatant was obtained using a 10 kDa Amicon and used for a fatty acid hydrolysis reaction.

As a result, as can be seen in Figures 3 to 4, it was confirmed that the production amount of PLA2 is the highest in BL21star (DE3).

Through this, BL21star (DE3) was selected as E. coli for constructing a transformant, and the following experiment was performed.

Example 3: Production of PLA2 enzyme derived from Streptomyces violaceoruber based on Escherichia coli

Example 3-1: Construction of a recombinant expression vector containing SvPLA2 and an E. coli transformant

To make SvPLA2 produced in E. coli and secreted to the outside of cells, a transformant was prepared.

More specifically, by inserting the amplified SvPLA2 enzyme gene into the plasmid vector pET-26b(+), a pET26b(+)-SvPLA2 vector containing the SvPLA2 enzyme gene that can be secreted into the cytoplasm of E. coli was constructed, and the plasmid vector pET26b (+) contains the N-terminal PelB signal sequence that causes the protein produced in the cell to be secreted into the periplasm. pET26b(+) vector was processed with restriction enzymes Msc I and Xho I to obtain fragments, and primers of 5'-GGCGTGGCCATGGCCCCCGCGGAC-3' (SEQ ID NO: 9, Msc I) and 3'-CCGCTCGAGGCCGAAGATCTTGACGG-5' (SEQ ID NO: 10, Xho I) The resulting SvPLA2 gene was amplified, and the amplified fragment was purified using a PCR purification kit. The purified PCR product was inserted into a plasmid vector using T4 DNA ligase. Thereafter, the expression vector pPICZαA-SvPLA2 prepared by the above method was used to transform E. coli BL21 star (DE3), and the transformed cells were grown in LB medium containing 100 μg/mL kanamycin.

Example 3-2: E. coli-based SvPLA2 production and activity

Fatty acid hydrolysis was performed using the enzyme produced from the transformant. Its experimental method is as described in Example 2.

As a result, as can be seen in Figure 5, it was confirmed that 22.8 mM (6.4 g/L) of linoleic acid was produced as a result of the reaction.

Through this, it was confirmed that E. coli-based SvPLA2 converts L-α-phosphatidylcholine by 76%, and it was confirmed that the production amount was 42% higher than that of the enzyme produced based on Pichia pastoris.

Example 4: Sciscionella marina-derived PLA2 (SmPLA2) enzyme production

Example 4-1: Construction of recombinant expression vector and E. coli transformant containing PLA2 derived from Sciscionella marina

In the present invention, PLA2 (SmPLA2) derived from Sciscionella marina, a marine microorganism, was newly discovered, which has a sequence identity of about 56% with SvPLA2. In order to produce a transformant in which the SmPLA2 is produced in E. coli and secreted to the outside of the cell, the amplified SmPLA2 enzyme gene was inserted into the plasmid vector pET-26b(+), and the pET26b(+)-SmPLA2 vector containing the SmPLA2 enzyme gene Was produced.

More specifically, SmPLA2 was synthesized by Cosmo Genetech (Seoul, Korea) based on the sequence, and the plasmid vector pET26b(+) is the N-terminal PelB signal sequence that allows the protein produced in the cell to be secreted into the periplasm. And the C-terminal his tag for purification, and primers of 5'-CCGGCGATGGCCATGGAGAGCATCGAAAGCATCAC-3' (SEQ ID NO: 11, NcoI) and 3'-GGTGGTGGTGCTCGAGACTGCCGAATTTGCGGAC-5' (SEQ ID NO: 12, Xho I) were prepared and SmPLA2 The gene was amplified. Thereafter, a fragment of DNA was obtained using a restriction enzyme corresponding to the underlined portion of the sequence and inserted into the plasmid vector pET26b(+) through in-fusion cloning. The expression vector pET26b(+)-SmPLA2 prepared by the above method was used to transform E. coli BL21 star (DE3), and the transformed cells were grown in LB medium containing 100 μg/mL kanamycin.

Example 4-2: E. coli-based SmPLA2 production

For the fatty acid hydrolysis reaction of the enzyme produced in the transformant, recombinant E. coli expressing the SmPLA2 gene was cultured in Resenberg's medium. 0.2 mM IPTG was added to the culture solution at OD600 = 1.0, and expression was induced at 25° C. and 200 rpm for 24 hours, and the culture solution was centrifuged to obtain a supernatant. Thereafter, a 10-fold concentrated supernatant was obtained using a 10 kDa Amicon to produce an SmPLA2 enzyme containing SEQ ID NO: 1 to be used in a fatty acid hydrolysis reaction.

Example 4-3: Comparison of SmPLA2 enzyme activity

In order to confirm the activity of the novel SmPLA2 enzyme, the bioconversion activity with SvPLA2 was compared.

More specifically, for fatty acid hydrolysis of enzymes produced in transformants, recombinant E. coli was cultured in Resenberg medium, 0.2 mM IPTG was added to the culture solution at OD600 = 1.0, and expressed for 24 hours at 25° C. and 200 rpm. Was induced, and the culture solution was centrifuged to obtain a supernatant. Thereafter, a 10-fold concentrated supernatant was obtained using a 10 kDa Amicon and used for a fatty acid hydrolysis reaction. 14 substrates available for the actual process? The activity of hydrolyzing soybean-derived lecithin to lysolecithin and linoleic acid was measured using a 23% low-purity substrate 62.2 g/L L-?-phosphatidylcholine (Sigma-Aldrich).

As a result, as can be seen in Figure 6, SvPLA2 was produced as a result of the reaction of 48 mM (13.5 g/L) linoleic acid, and SmPLA2 was produced as a result of the reaction of 37 mM (10.4 g/L) linoleic acid. Confirmed.

Through this, it was confirmed that the amount of hydrolysis production using SvPLA2 and SmPLA2 enzymes was similar.

Example 5: Characterization of SvPLA2 and SmPLA2

Example 5-1: Analysis of optimal pH and temperature of SvPLA2

The two main variables of biotransformation reactions for measuring enzyme activity are temperature and pH. For the characterization of SvPLA2, the optimum pH and temperature were confirmed.

More specifically, the comparison of the bioconversion reaction according to the pH is as described in the performance of the low-purity substrate carried out in Example 4-3.

As a result, as can be seen in FIG. 7, it was confirmed that the activity was relatively high at pH 7.0-7.5, and it was confirmed that the highest activity was shown at pH 7.5 with a slight difference.

In addition, the comparison of the bioconversion reaction according to temperature was carried out at pH 7.5, temperatures 40, 50, 60, and 70°C in the same manner as described above.

As a result, as can be seen in Fig. 8, it exhibited the highest activity at 50 ℃.

Through this, it was confirmed that the optimum pH was 7.5 and the temperature was 50°C.

Example 5-2: Analysis of optimal pH and temperature of SmPLA2

In order to determine the characteristics of SmPLA2, the optimum reaction pH was determined in the same manner as in Example 5-1, and the optimum reaction temperature was determined after applying the optimum pH.

As a result, as can be seen in Figures 9 to 10, it was confirmed the highest activity at pH 7.5 and 50 ℃.

Through this, it was confirmed that the optimal reaction conditions for SmPLA2 were the same as the optimal reaction conditions for SvPLA2, and that the optimal activity conditions were the same.

Example 6: Comparison of biotransformation activity under optimal conditions

The biotransformation activities of SvPLA2 and SmPLA2 in the optimal conditions found in Example 5 were compared. The biotransformation activity experiment is as described above.

As a result, as can be seen in FIG. 11, it was confirmed that 45 mM (12.6 g/L) of linoleic acid was produced as a result of SvPLA2, and 38 mM (10.7 g/L) of linoleic acid was produced as a result of the reaction. It was confirmed that it was created.

Through this, it was confirmed that the yield obtained by hydrolyzing soybean-derived lecithin to lysolecithin and linoleic acid through SvPLA2 and SmPLA2 was similar, and that the characteristics of SvPLA2 and SmPLA2 were similar.

Example 7: Evaluation of heat resistance of SmPLA2

In order to evaluate the heat resistance of SmPLA2, a heat resistance comparison experiment was performed with SvPLA2 known in the past. The heat resistance experiment of the enzyme was carried out in a flask in a shaking incubator at 180 rpm, 50°C and 60°C, and 50 mM Tris-HCl buffer (pH 7.5) and 0.66 g/L calcium chloride aqueous solution were added to the flask. It was incubated with a concentrated supernatant of 10% volume concentration.

More specifically, the cultivation was performed for 0, 1, 3, and 5 hours, and samples of each time period were mixed with a substrate prepared at the same temperature, and 14? Of Example 2-3? The reaction was carried out as in the example of 23% low purity substrate.

As a result, as can be seen in Figure 12, SvPLA2 after 1 hour incubation at 50 ℃, the activity rapidly decreased to 20%, but SmPLA2 was confirmed that the activity is maintained about 80% even after 5 hours incubation at 50 ℃.

In addition, it was confirmed that SmPLA2 maintained 80% activity similar to that at 50°C even after 5 hours incubation at 60°C.

Through this, it was confirmed that SmPLA2 was remarkably excellent in heat resistance compared to SvPLA2, and it was confirmed that structural stability due to heat was increased.

Example 8: Marinoactinospora thermotolerans-derived PLA2 (MtPLA2) enzyme production

Example 8-1: Marinoactinospora thermotolerans-derived PLA2 containing recombinant expression vector and E. coli transformant construction

In the present invention, PLA2 (MtPLA2) derived from Marinoactinospora thermotolerans, a marine microorganism, was newly discovered, which has a sequence identity of about 45% with SvPLA2. In order to produce a transformant in which the MtPLA2 is produced in E. coli and secreted to the outside of the cell, the amplified MtPLA2 enzyme gene was inserted into the plasmid vector pET-26b(+), and pET26b(+)-MtPLA2 vector containing the MtPLA2 enzyme gene Was produced.

More specifically, MtPLA2 was synthesized by Cosmo Genetech (Seoul, Korea) based on the sequence, and the plasmid vector pET26b(+) is the N-terminal PelB signal sequence that allows the protein produced in the cell to be secreted into the periplasm. And a C-terminal his tag for purification, and primers of 5'-CCGGCGATGGCCATGGCGCTCACCCCCGAGCA-3' (SEQ ID NO: 13, NcoI) and 3'-GGTGGTGGTGCTCGAGCCCGACCGCGTGGCG-5' (SEQ ID NO: 14, Xho I) were prepared to make MtPLA2 The gene was amplified. Thereafter, a fragment of DNA was obtained using a restriction enzyme corresponding to the underlined portion of the sequence, and inserted into the plasmid vector pET26b(+) through in-fusion cloning. The expression vector pET26b(+)-MtPLA2 prepared by the above method was used to transform E. coli BL21 star (DE3), and the transformed cells were grown in LB medium containing 100 μg/mL kanamycin.

Example 8-2: E. coli-based MtPLA2 production

For the fatty acid hydrolysis reaction of the enzyme produced in the transformant, recombinant E. coli expressing the MtPLA2 gene was cultured in Resenberg's medium. At OD600 = 1.0, 0.2 mM IPTG was added to the culture solution, and expression was induced at 25°C and 200 rpm for 24 hours, and the culture solution was centrifuged to obtain a supernatant. Thereafter, a 10-fold concentrated supernatant was obtained using a 10 kDa Amicon to produce an MtPLA2 enzyme containing an amino acid sequence (SEQ ID NO: 2) to be used in a fatty acid hydrolysis reaction.

Example 9: Characterization of MtPLA2

In order to determine the characteristics of MtPLA2, the optimum reaction pH was determined in the same manner as in Example 5, and the optimum reaction temperature was determined after applying the optimum pH.

As a result, as can be seen in Figures 13 to 14, the highest activity was confirmed at pH 7.5 and 70 ℃.

Through this, compared to the optimal reaction temperature of SvPLA2 and SmPLA2 is 50 ℃, it was seen that the optimum reaction temperature of MtPLA2 is 70 ℃, it was confirmed that MtPLA2 shows high activity at high temperature compared to other derived PLA2.

Example 10: Evaluation of heat resistance of MtPLA2

In order to evaluate the heat resistance of MtPLA2, a comparison experiment was performed with SvPLA2 known in the past. The experimental method is as described in Example 7.

As a result, as can be seen in FIG. 15, SvPLA2 was rapidly reduced to 20% after 1 hour of incubation at 50°C, but MtPLA2 was maintained at more than 60% after 1 hour at 50°C, and even after 5 hours, 40 It was confirmed that more than% activity was maintained.

In addition, it was confirmed that MtPLA2 maintained 50% activity even after 3 hours incubation at 60°C.

Through this, it was confirmed that MtPLA2 was remarkably excellent in heat resistance compared to the previously known SvPLA2, and it was confirmed that structural stability due to heat was increased.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed as including all changes or modified forms derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts thereof.

<110> Ewha University-Industry Collaboration Foundation Chung-Ang University Industry-Academy Cooperation Foundation <120> Novel thermostable phospholipase A2 and process for preparing phospholipase A2 <130> KPA191196-KR <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> recombinant protein <400> 1 Met Asn Thr Thr Gly Val Arg Arg Gly Ala Leu Ser Ser Ala Ile Val 1 5 10 15 Ala Ile Thr Ile Ala Leu Gly Ala Gly Thr Ala Gly Ala Glu Ser Ile 20 25 30 Glu Ser Ile Thr Asp Asn Tyr Leu Phe Ser Thr Ser Pro Ser Gln Phe 35 40 45 Glu Lys Val Arg Asp Glu Arg Pro His Ala Asp Lys Leu Asp Trp Ser 50 55 60 Ser Asp Ser Cys Ser Trp Ala Pro Asp Lys Pro Val Gly Phe Asp Phe 65 70 75 80 Asp Pro Ala Cys His Arg His Asp Phe Gly Tyr Arg Asn Tyr Lys Lys 85 90 95 Gln Ser Arg Phe Asp Asp Thr Ser Lys Lys Arg Ile Asp Asp Asn Phe 100 105 110 Tyr Ser Asp Leu Lys Gly Ile Cys His Gly Asn Gly Ser Cys Asn Ala 115 120 125 Leu Ala Trp Thr Tyr Tyr Gln Ala Val Arg Lys Phe Gly Ser 130 135 140 <210> 2 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> recombinant protein <400> 2 Met Arg Leu Val Ile Arg Arg Ala Val Thr Ala Val Ala Thr Ala Ala 1 5 10 15 Ala Ala Val Leu Leu Thr Leu Pro Thr Ala Thr Gly Ala His Ala Ala 20 25 30 Leu Thr Pro Glu Gln Val Arg Gly Val Thr Asp Thr Tyr Leu Phe Ala 35 40 45 Thr Pro Leu Ala Glu Phe Asp Ala Ile Arg Asp Gln Arg Pro His Ala 50 55 60 Asp Gln Leu Val Trp Asp Ser Asp Gly Cys Ser Trp Ser Pro Asp Glu 65 70 75 80 Pro Phe Gly Tyr Glu Phe Leu Ser Ser Cys Glu Arg His Asp Phe Gly 85 90 95 Tyr Arg Asn Tyr Lys Gln Gln Ser Arg Phe Thr Glu Glu Ala Arg Leu 100 105 110 Arg Ile Asp Asn Gln Phe Arg Thr Asp Met Tyr Ser Val Cys Gly Gly 115 120 125 Asn Val Leu Cys Arg Gly Val Ala Val Ile Tyr Tyr Asn Ala Val Arg 130 135 140 Glu Phe Gly Gly Ser Gly Val Ser Thr Ala Glu Ala Leu Glu Arg Ala 145 150 155 160 Asn Ala Val Glu Arg Ala Glu Ser Tyr Leu Ala Arg His Ala Val Gly 165 170 175 <210> 3 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 3 gagagcatcg aaagcatcac cgacaactac ctgttcagca cctcgccgag ccagttcgag 60 aaagtccgcg acgagagacc gcacgccgac aagctcgact ggtccagcga ttcctgctcc 120 tgggccccgg acaaaccggt cggcttcgac ttcgatccgg cctgtcaccg ccacgatttc 180 ggctaccgga actacaagaa gcagagccgg ttcgacgaca ccagcaaaaa gcgcatcgac 240 gacaacttct acagcgatct caagggaatc tgccacggca acggcagctg caacgcgctc 300 gcctggactt actaccaagc ggtccgcaaa ttcggcagt 339 <210> 4 <211> 435 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 4 gcgctcaccc ccgagcaggt gcgcggcgtc accgacacct acctcttcgc gacgccgctg 60 gcggagttcg acgcgatccg cgaccagcgt ccgcacgccg accagctcgt ctgggactcc 120 gacgggtgca gctggtcccc ggacgagccc ttcgggtacg agttcctgtc cagctgcgag 180 cgccacgact tcggctaccg caattacaaa cagcagtccc ggttcaccga agaggcgcgg 240 ctgcgtatcg acaaccagtt ccgcaccgac atgtactcgg tgtgcggcgg caacgtgctg 300 tgccgtgggg tcgccgtcat ctactacaac gccgtgcggg agttcggcgg gtcgggggtc 360 agcacggccg aggccctgga gcgggcgaac gcggtcgagc gcgccgagtc ctacctcgcg 420 cgccacgcgg tcggg 435 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 5 gactagtgct cctgcagaca aacc 24 <210> 6 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 6 ctagctagct ccaaaaatct taactgcttg ataataagt 39 <210> 7 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 7 ctagctagcc atcatcatca tcatcattga gtttgt 36 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 8 gactagtagc ttcagcctct cttttctc 28 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 9 ggcgtggcca tggcccccgc ggac 24 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 10 ccgctcgagg ccgaagatct tgacgg 26 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 11 ccggcgatgg ccatggagag catcgaaagc atcac 35 <210> 12 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 12 ggtggtggtg ctcgagactg ccgaatttgc ggac 34 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 13 ccggcgatgg ccatggcgct cacccccgag ca 32 <210> 14 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 14 ggtggtggtg ctcgagcccg accgcgtggc g 31

Claims (14)

Culturing a recombinant E. coli expressing a polynucleotide encoding a phospholipase A2 (PLA2) enzyme comprising the amino acid sequence of SEQ ID NO: 1 to obtain a phospholipase A2 enzyme; And
It comprises the step of reacting the phospholipase A2 enzyme and lecithin to hydrolyze into lysolecithin,
The phospholipase A2 enzyme is a method of hydrolyzing lecithin to lysolecithin using a phospholipase A2 enzyme, characterized in that it has heat resistance at 50 ℃ to 60 ℃.
The method of claim 1, wherein the PLA2 enzyme comprising the amino acid sequence of SEQ ID NO: 1 is derived from Sciscionella marina.
The method of claim 1, wherein the phospholipase A2 enzyme and lecithin are reacted at a temperature of 50°C to 60°C.
The method of claim 1, wherein linoleic acid is produced as a product of the hydrolysis.
The method of claim 1, wherein the PLA2 enzyme comprising the amino acid of SEQ ID NO: 1 has an optimal biotransformation activity at a pH of 7.5 and a temperature of 50°C.
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