KR20080093586A - Recombinant plasmid vector, Seratia Masson genus recombinant strain transformed with recombinant plasmid vector and lipase production method using the same - Google Patents

Abstract

The present invention transforms the DNA encoding the tliDEF ABC transporter and lipase (tliA) extracted from the Pseudomonas fluorescence strain to the Serratia masons strain using a plasmid vector to produce a large amount of lipase of higher activity than the existing strain. The system was built. Through this, the lipase can be obtained in the activated form instead of the inclusion body using the ABC-type protein secretion carrier, and since the lipase is discharged in vitro, the lipase can be obtained without grinding the original strain, which is very economical and yields. Is also high.

Furthermore, the production efficiency of lipase increases rapidly when the plasmid vector derived from a specific plasmid vector is transformed into a specific strain of Serratia matisse.

Description

Recombinant PLASMID VECTOR, SERRATIA MARCESCENS RECOMBINANT BACTERIA TRANSFORMED BY THE RECOMBINANT PLASMID VECTOR AND LIPASE MANUFACTURING METHED USING THEREOF}

1A-1D are genetic maps of the recombinant plasmid vectors of the present invention.

Figure 2 is a photograph showing a clear ring that occurs when the recombinant Serratia genus strain of the present invention incubated in an LBT plate.

3A and 3B are graphs showing cell growth and lipase activity in 100 ml of LB medium of selected Seratia recombinant strains.

4 is a diagram showing the results of SDS-PAGE electrophoresis of selected Seratia recombinant strains.

5 is a graph showing cell growth and lipase activity when the recombinant plasmid of the present invention is introduced and cultured in recombinant E. coli.

6 is a graph showing cell growth and lipase activity when the recombinant plasmid of the present invention is introduced and cultured in Pseudomonas fluorescence.

The present invention relates to a recombinant plasmid vector, a recombinant strain of the genus Serratia masons transformed with a recombinant plasmid vector, and a method for producing a lipase using the same. More specifically, a method for producing a lipase capable of producing a lipase having higher activity than a conventional strain. And a recombinant plasmid vector used therein, and a Seratia Mason genus recombinant strain transformed with the recombinant vector.

In the past decades, with the development of biotechnology, we have discovered and characterized various genes from various organisms, from microorganisms to humans. Once the sequencing has been determined, the work of putting these genes back into the host cell to make the desired protein Has been made. As a representative host used for the introduction of genes, Escherichia coli has been used for a long time as a laboratory research tool, which is more researched than other hosts, is convenient to use, and has been modified in various ways to suit the purpose. There are many E. coli bacteria, and they are useful not only for research but also for the production of useful proteins for industrial mass production.E. Coli accumulates 20-50% of the protein of foreign genes, so it can be obtained in excess. However, when the foreign gene is expressed in Escherichia coli, the protein is often made of an inactive inclusion body rather than the active form.

In order to solve this problem, studies have been conducted to secrete proteins expressed in cells out of cells. By secreting the expressed protein out of the cell, the protein produced in the cell can be secreted out before it accumulates so that it does not form inclusion bodies. The most ideal for producing proteins using microorganisms is to obtain the desired protein directly from the microbial culture when the protein expressed in the cells is secreted out of the cell to remove only the microorganism from the microbial culture.

One of the methods of protein secretion is the ABC-type protein secretion carrier (ABC transporter), which is a ABC transporter because it has a cassette that binds to ATP because it secretes proteins while decomposing ATP. Protein secreted by this secretion pathway has a carboxy terminal targeting signal (C-terminal targeting signal), which is recognized by the ABC-type protein secretion carrier, a secretagogue protein, and secreted out of the cell. The secretion of proteins by the ABC-type protein secretion carrier system is made by the ABC-type protein secretion carrier proteins present in the cell membrane. When the ABC-type protein secretion carrier system is used, both Gram-positive bacteria and Gram-negative bacteria pass through the membrane at once. You can do it. That is, in the case of Gram-negative bacteria, the protein secreted with one of the ABC-type protein secretion carriers is partially fused when passing through the inner and outer membranes of the gram-negative bacteria so that the protein passes through two biological membranes simultaneously. Therefore, in the case of Gram-negative bacteria, the secretion system by the ABC-type protein secretion carrier can secrete the protein out of the cell in a simple and effective manner.

So far, the case studies on the secretion promoter protein type ABC secretion carrier and the lipase secreted by it are as follows.

TABLE 1

Lipase enzymes are mainly used in the food industry, feed industry, alternative energy industry, pharmaceutical industry and detergent industry, and the main producing microorganisms are bacteria, yeast and fungi. There are many varieties, and from an industrial point of view, the enzyme production mainly used yeast Candida and bacteria Bulcolderia. Lipases have been applied to various fields, and new lipase producing strains have been studied to meet such demands.

Problems in the production of lipases are difficult in process development, but there are problems in securing excellent strains. Since the action and activity of the produced enzymes are very different depending on the types of microorganisms producing lipases, there is a need for the discovery of new microorganisms with lipase-producing ability and research on the production of enzymes using them is very important.

The present invention has been made to solve the above-mentioned problems, an object of the present invention is to transform the Seratia Masons strain by transforming the Gram-negative ABC-type protein secretion carrier and lipase DNA to Serratia Masons strain using a plasmid vector It is to provide a new extracellular lipase production system that makes a strain, and produces a lipase having higher activity than the existing strain from the recombinant strain.

Recombinant plasmid vector for solving the technical problem of the present invention is characterized in that the gene encoding the ABC-type protein secretion carrier and lipase derived from Gram-negative bacteria was introduced into the plasmid vector.

The plasmid vector which can be used at this time is E. coli plasmid, preferably any one plasmid vector selected from the group consisting of pACYC184, pKK223-3, pDSK519 and pHCE can be used.

The Gram-negative bacterium is preferably of the genus Pseudomonas fluorescens , more preferably, the gene encoding the ABC-type protein secretion carrier is tliDEF, and the gene encoding the lipase may use DNA including tliA. have.

The recombinant plasmid vector preferably has a genetic map represented by any one of FIGS. 1A, 1B, 1C and 1D.

Seratia Mason genus recombinant strain according to another embodiment of the present invention is transformed with the above-described recombinant plasmid vector, in this case, the strains of the genus Serratia Masons preferably SM2216 (KCTC2216), SM2356 (KCTC2356), SM2516 (KCTC2516), Either SM2798 (KCTC2798) or SM2801 (KCTC2801) can be used.

Lipase production method by a gene recombination method according to another embodiment of the present invention, 1) preparing a recombinant plasmid vector in which a tliDEF ABC transporter derived from Pseudomonas fluorsonson strain and a DNA fragment encoding a lipase is introduced, 2 1) preparing a recombinant strain of Seratia masons genus transformed with the recombinant plasmid vector, 3) inducing extracellular secretion of lipase by culturing the transformed Serratia masons genotype strain in a medium; And 4) recovering lipase from the culture of the transformed recombinant strain.

The recombinant plasmid vector preferably has a genetic map represented by any one of Figs. 1A, 1B, 1C and 1D, and the genus strains of Serratia masons are SM2216 (KCTC2216), SM2356 (KCTC2356), SM2516 (KCTC2516), One of SM2798 (KCTC2798) and SM2801 (KCTC2801).

Said Seratia Massons recombinant strain is preferably a strain transformed with a recombinant plasmid vector derived from pKK223-3 with the original strain of SM2356 (KCTC2356), and transformed with a recombinant plasmid vector derived from pKK223-3 with the original strain of SM2798 (KCTC2798). Any one selected from the group consisting of a strain transformed with a recombinant plasmid vector derived from pDSK519, and a strain transformed with a recombinant plasmid vector derived from pDSK519 and a native strain is SM2356 (KCTC2356). The efficiency of the strain is the best.

Hereinafter, the present invention will be described in more detail.

The present invention transforms Seratia Masons strain by transforming ABC type protein secretion carrier and lipase DNA of Gram-negative bacterium into plasmid vector to make Seratia Masons recombination strain, and from this recombinant strain lipase having higher activity than existing strain It is to provide a new extracellular lipase production system to produce.

To this end, there is provided a recombinant plasmid vector having a gene encoding an ABC-type protein secretion carrier derived from Gram-negative bacteria and a lipase as a plasmid vector.

At this time, the plasmid vector which can be used is E. coli plasmid vector, and more preferably any one plasmid vector selected from the group consisting of pACYC184, pKK223-3, pDSK519 and pHCE which is usually used as a plasmid vector for E. coli can be used. have.

On the other hand, the Gram-negative bacteria that can be applied in the present invention is preferably a strain of Pseudomonas fluorescens ( Pseudomonas fluorescens ), preferably Pseudomonas comprising a gene encoding a ABC-type protein secretion carrier and a gene encoding a lipase If the strain of the genus Pseudomonas fluorescens ( Pseudomonas fluorescens ) can be used without limitation, but more preferably Pseudomonas fluorescens SIK W1 ( Pseudomonas fluorescens SIK W1, KCTC2689P) can be used. The gene encoding the ABC-type protein secretion carrier derived from the Pseudomonas fluorescens genus strain is preferably known tliDEF (GenBank ID: AAD09853, AAD09854, AAD09855), and the gene encoding the lipase is tliA ( GenBank ID: AAD09856) can be used.

The vectors of the recombinant plasmids constructed in this configuration were pTliDEFA-184 (tliDEFA introduced in pACYC184), pTliDEFA-223 (tliDEFA introduced in pKK223-3), pTliDEFA-519 (tliDEFA introduced in pDSK519), pTliDEFA-HCE ( tliDEFA is introduced into pHCE), which consists of a genetic map represented by FIGS. 1A, 1B, 1C, and 1D, respectively, and can be prepared by a conventional method for preparing a recombinant plasmid vector.

In a second aspect of the present invention, there is provided a Seratia Mason genus recombinant strain transformed with the recombinant plasmid vector of the above configuration. The strains of Serratia Masons used in the present invention were strains of the genus Serratia, which were distributed from the Korea Biotechnology Research Institute Gene Bank (KCTC). More specifically, SM2216 (KCTC2216), SM2356 (KCTC2356), SM2516 (KCTC2516), SM2798 (KCTC2798) and SM2801 (KCTC2801) strains were used. Recombinant strains were named using plasmids and native strains. For example, a recombinant strain made by transforming the pTliDEFA-HCE plasmid into the SM2216 progeny strain was designated as HCE-2216, and a recombinant strain made by transforming the pTliDEFA-223 plasmid into the SM2516 progeny strain, respectively.

The lipase production method according to the third aspect of the present invention comprises the steps of: 1) preparing a recombinant plasmid vector having a DNA fragment encoding a lipase and a tliDEF ABC transporter derived from Pseudomonas fluorescence strain, and 2) the recombinant plasmid vector. Preparing a transformed strain of Serratia masons, 3) inducing extracellular secretion of lipase by culturing the transformed Serratia masons recombinant strain in a medium, and 4) the transformed recombinant Recovering lipase from the culture of the strain.

Step 1) is a step for preparing a recombinant plasmid vector, which means the recombinant plasmid vector described in the first embodiment of the present invention, and is prepared by a method for preparing a conventional recombinant plasmid vector.

Step 2) is a step of preparing a Seratia Mason genus recombinant strain transformed with the recombinant plasmid vector prepared in step 1), which means the Seratia Mason genus recombinant strain described in the second form of the present invention , The Seratia Mason genus recombinant strain may be prepared by a method of transforming the original strain through a conventional recombinant plasmid vector, and more specifically, it may be used, such as electric shock, but is not limited thereto.

The present invention may further comprise the step of selecting the Seratia Mason genus recombinant strain prepared in step 2). In the recombinant strain selection step, the supernatant was recovered by culturing the recombinant strain in a test tube and centrifugation, and the recombinant strain having high lipase activity was selected through lipase activity investigation of the supernatant.

Step 3) is a step of inducing the extracellular secretion of lipase by culturing the transformed Seratia Mason genus recombinant strain prepared in step 2) in a medium, the culture conditions and the like described in the Examples described later It may be cultured by, but is not limited thereto.

Step 4) is a step of recovering and purifying the lipase secreted onto the culture medium through the extracellular secretion in step 3) to recover and purify the lipase through centrifugation. When centrifugation, the recombinant strain cell is settled, the supernatant is formed into the culture solution, and since the lipase expressed from the recombinant strain is dissolved in the supernatant, it is recovered and purified by a conventional method.

On the other hand, the recombinant strain of the genus Serratia masons is preferably a strain transformed with a recombinant plasmid vector derived from pKK223-3 with the original strain of SM2356 (KCTC2356), and a recombinant plasmid vector derived from pKK223-3 with the original strain of SM2798 (KCTC2798). Any one selected from the group consisting of a transformed strain, a strain transformed with a recombinant plasmid vector derived from pDSK519 with a native strain of SM2356 (KCTC2356) and a strain transformed with a recombinant plasmid vector derived from pDSK519 with a native strain of SM216 (KCTC2516). The efficiency of the recombinant strain is the best and can be confirmed in Example 5 to be described later.

Hereinafter, the present invention will be described in detail by way of examples. The following examples are merely illustrative of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1 Construction of Recombinant Plasmid Vectors

One. pTliDEFA Made of -223

pTliDEFA-223 is a recombinant plasmid vector in which tliDEFA was introduced into pKK223-3, a common plasmid vector for Escherichia coli, and used a polymerase chain reaction to secure tliDEFA gene, wherein tliDEFA, prtA (protein hydrolase gene), pTOTAL (protein: Ahn, JH et al., J. Bacteriol., 181: 1847-1852, 1999) in which inh (protein hydrolase inhibitor gene) was linked to the pUC19 vector was prepared as a template for polymerase chain reaction. The primers used were 5'-GCCGAATTCATGTCGTTTGGTTCAAGGAAG-3 'as a forward primer and 5'-AGCAAGCTTATGAACCGCCGATAATCCCGTC-3' as a reverse primer, EcoRI recognition site in the forward primer and HindIII recognition site in the reverse primer. . The amplified gene was isolated by agar gel electrophoresis, and the amplified separated DNA fragment was digested with EcoRI and HindIII, and then conjugated with pKK223-3 digested with the same restriction enzyme to prepare a recombinant plasmid vector represented by FIG. 1A. It was.

2. pTliDEFA -184 Made

pTliDEFA-184 is a recombinant plasmid vector in which tliDEFA was introduced into pACYC184, which is a common E. coli plasmid vector. The polymerase chain reaction was used to secure the tliDEFA gene, and pTOTAL was prepared as a template for polymerase chain reaction. The primers used were 5'-ATTGATATCCTCCTGATCGGGGGTGCGGGC-3 'and 5'-ATTGATATCATGAACCGCCGATAATCCCGTC-3' as a reverse primer, EcoRV recognition site in the forward primer, and XhoI recognition site in the reverse primer. . The amplified gene was isolated by agar gel electrophoresis, and the amplified separated DNA fragment was digested with EcoRV and XhoI, and then conjugated with pACYC184 digested with EcoRV and SalI to prepare a recombinant plasmid shown in FIG. 1B.

3. Fabrication of pTliDEFA-519

pTliDEFA-519 is a recombinant plasmid vector in which tliDEFA was introduced into pDSK519, a common E. coli plasmid vector. The polymerase chain reaction was used to secure the tliDEFA gene, and pTOTAL was prepared as a template for polymerase chain reaction. The primers used were 5'-GCCTCTAGAATGTCGTTTGGTTCAAGGAAG-3 'as a forward primer and 5'-ATTGAGCTCATGAACCGCCGATAATCCCGTC-3' as a reverse primer, XbaI recognition site in the forward primer, and SacI recognition site in the reverse primer. . The amplified gene was isolated by agar gel electrophoresis, and the amplified separated DNA fragment was digested with XbaI and SacI, and then conjugated with pDSK519 digested with the same restriction enzyme to prepare a recombinant plasmid represented by FIG. 1C.

4. pTliDEFA - HCE Made of

pTliDEFA-HCE is a plasmid vector in which tliDEFA was introduced into pHCE, a common E. coli plasmid vector. The polymerase chain reaction was used to secure the tliDEFA gene, and pTOTAL was prepared as a template for polymerase chain reaction. The primers used were 5'-GCCCATATGATGTCGTTTGGTTCAAGGAAG-3 'as a forward primer and 5'-ATTGAGCTCATGAACCGCCGATAATCCCGTC-3' as a reverse primer, NdeI recognition site in the forward primer, and XbaI recognition site in the reverse primer. . The amplified gene was separated by agar gel electrophoresis, and the amplified separated DNA fragment was digested with NdeI and XbaI, and then conjugated with pHCE digested with the same restriction enzyme to prepare a recombinant plasmid shown in FIG. 1D.

Example 2 Preparation of Seratia Masons Recombinant Strain

The four recombinant plasmid vectors prepared in Example 1 were prepared using five strains of Serratia Masons [SM2216 (KCTC2216), SM2356 (KCTC2356), SM2516 (KCTC2516), SM2798 (KCTC2798), and SM2801 (KCTC2801). It was prepared in the form of cells for electrical transformation. Cells for electrical transformation were prepared by the following process.

1) Probiotics were plated on LB plates (1% trypton, 0.5% yeast extract, 1% NaCl, 1.5% agar) and incubated in a 25 ° C. incubator for 24 hours.

2) Colonies grown on LB plates were inoculated into 200 ml of LB medium and incubated at 25 rpm shaking incubator at 150 rpm until the OD was 0.6 (measured at 600 nm UV).

3) The culture medium and the original strain cells were separated by centrifugation, and the culture medium was discarded and washed with 30 ml of 10% glycerol.

4) 10% glycerol and prokaryotic cells were separated by centrifugation, and the separated 10% glycerol was discarded.

5) Repeat the above 3), 4) process three times.

6. The washed prokaryotic cells obtained through centrifugation were released in 3 ml of 10% Glycerol.

When the four recombinant plasmid vectors prepared in Example 1 were introduced into microbial cells by electric shock, 80 μl of the transformed cells and 5ul of plasmid were placed in an electroshock transformation cuvette, and a 1.8 kV voltage was applied for 5 ms. Add 1 ml of SOC medium (2% tryptone, 0.5% yeast extract, 0.05% NaCl, 0.0186% KCl, 0.095% MgCl2, 0.36% glucose) and incubate for 1 hour in a 37 ° C incubator. Was prepared. The prepared recombinant strain was plated on LB plate (1% trypton, 0.5% yeast extract, 1% NaCl, 1.5% agar) and incubated at 25 ℃.

Example 3 Lipase Activity Measurement

The lipase activity was measured by pNP (p-Nitrophenol) assay. When 10 mM pNPP (p-Nitrophenyl palmitate), ethanol, and 50 mM Tris-HCl (pH 8.0) were added to the reaction solution in a ratio of 1: 4: 95, the microbial culture supernatant was added and reacted at 45 ° C. The lipase of the microbial culture supernatant decomposed pNPP. It is divided into pNP and palmitic acid. The concentration of pNP generated at this time was measured by absorbance at 405 nm ultraviolet spectrophotometer to measure lipase activity. At this time, one unit (unit) was defined as 1umol pNP produced per minute.

Example 4 Selection of Recombinant Strains

In order to select recombinant strains capable of producing high activity lipases among the 20 recombinant strains prepared in Example 2, 5 ml LB broth (1% tryptophan, 0.5% yeast extract, 1% NaCl) liquid in vitro. The culture was carried out.

After incubating 5 original strains and 20 recombinant strains in an LB plate to which 0.8% tributirin was added (see FIG. 2), a colony showing a transparent ring was selected and inoculated into a test tube containing 5 ml LB broth to incubate at 25 ° C and 100 rpm. After culturing for 24 hours at lipase activity was measured and the results are shown in Table 2.

TABLE 2

As can be seen from Table 2, five recombinant strains with superior lipase activity were selected as compared to the original strain, and the selected recombinant strains were 184-2216, 223-2356, 223-2798, 519-2356. , 519-2516.

Example 5 Confirmation of Lipase Expression in Selected Recombinant Strains

In order to confirm the lipase expression of the five recombinant strains selected in Example 4, the culture was performed on 100 ml LB broth in a 250 ml Erlenmeyer flask. Five selection recombinant strains were grown on LBT plates and single colonies with clear rings were picked and inoculated into 250 ml Erlenmeyer flasks containing 100 ml of LB broth. Cell growth, lipase activity was measured while culturing the Erlenmeyer flask inoculated with the recombinant strain at 25 ° C. and 100 rpm, and the lipase was confirmed by SDS-PAGE. Cell growth was measured for absorbance in a 600 nm UV spectrophotometer, and lipase activity was measured using the lipase activity assay of <Example 3>. The concentration of Resolving gel of SDS-PAGE was 10%, and CBB (Coomassie brilliant blue G-250) was used as a staining reagent.

As a result of measuring lipase activity, four recombinant strains 223-2356, 223-2798, 519-2356, and 519-2516 except 184-2216 showed high lipase activity of up to 2000 ~ 2700 unit / L, and 184-2216. In the case of recombinant strains showed up to 532 units / L activity (see Figure 3). In addition, as a result of SDS-PAGE, lipase was confirmed in the section of about 50 kDa (see FIG. 4).

Example 5 Comparison with Escherichia Coli and Other Gram-Negative Strains

To confirm the efficiency of recombinant Serratia strains in lipase production using ABC-type protein secretion carriers, we compared the secretion capacity of Escherichia coli and Pseudomonas bacteria with host cells. As in the case of the Serratia strain, the same recombinant plasmid vectors as in Example 1 were introduced into E. coli JM109. After culturing Escherichia coli under the same conditions as in the Ceratia host cell culture, the culture supernatant was recovered to investigate the amount of lipase secretion. Even when the highest amount of lipase was produced from Escherichia coli host cells as shown in Figure 5, the secretion yield was very low compared to when using the Serratia strain. In conclusion, the lipase secretion produced by the ABC protein secretion carrier from Serratia genus was about 150 times higher than that of Escherichia coli. The comparison result of E. coli and Serratia strains showed the same result in the experiment using E. coli JM109 as well as various E. coli strains (eg XL10-gold, BL21, DH5a).

In order to verify the lipase production efficiency using the ABC-type protein secretion carrier from the Serratia strain, it was compared with Pseudomonas fluorescence, which is a natural original strain of TliA lipase applied in the examples of the present invention. Plasmid pTliDEFA-519 was introduced into the Pseudomonas fluorescence strain, and cultured in the same manner as in the culture conditions of Serratia host cells. The culture supernatant was recovered and examined for the amount of lipase secretion. As shown in FIG. 6, the lipase secreted by the ABC protein secretion carrier from the Pseudomonas fluorescence host cell was expressed by the ABC protein secretion carrier from the Serratia strain of the present invention. Only 35% of the time. Studies of lipases secreted from Pseudomonas strains have also been studied in other literature (Ahn et al., 2001), where even the strains with the highest lipase secretion efficiency showed significantly lower secretion yields than the Serratia strains of the present invention. .

In the present invention, the lipase can be obtained in an activated form rather than in an inclusion body form using the ABC-type protein secretion carrier. In addition, since the lipase is discharged to the outside of the original strain, the lipase can be obtained without grinding the original strain, which is very economical and yields are high.

Furthermore, when the plasmid vector derived from a specific plasmid vector was transformed into a specific strain of Serratia matisse, it was confirmed that the production efficiency of lipase increased rapidly.

As a result, the present invention constructs a production system for transforming seratiasons strains using tliDEF ABC transporter and lipase (tliA) DNA extracted from Pseudomonas fluorescence strains to produce higher active lipases than conventional strains. By doing so, it can be said that if the framework for mass production is prepared in the future, it has laid the foundation for mass production of high quality lipase.

Although the present invention has been described in detail only with respect to the embodiments described, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical spirit of the present invention, and such modifications and variations belong to the appended claims. .

Claims (12)

Recombinant plasmid vector, characterized in that the plasmid vector is introduced into the ABC-type protein secretion carrier and lipase gene derived from Gram-negative bacteria. The method of claim 1, The plasmid vector is the recombinant plasmid vector, characterized in that the E. coli plasmid vector. The method of claim 2, The recombinant plasmid vector of E. coli is any one selected from the group consisting of pACYC184, pKK223-3, pDSK519 and pHCE. The method of claim 1, The Gram-negative bacterium is Pseudomonas fluorescence fluorescens ) the recombinant plasmid vector, characterized in that the genus. The method of claim 1, The recombinant plasmid vector, wherein the gene encoding the ABC-type protein secretion carrier is tliDEF, and the gene encoding the lipase is tliA. The method of claim 1, The recombinant plasmid vector has a genetic map represented by any one of Figures 1a, 1b, 1c and 1d. The Seratia Mason genus recombinant strain transformed with the recombinant plasmid vector of any one of claims 1 to 6. The method of claim 7, wherein Said Seratia Masons genus strain is any one of SM2216 (KCTC2216), SM2356 (KCTC2356), SM2516 (KCTC2516), SM2798 (KCTC2798) and SM2801 (KCTC2801). In the lipase production method by genetic recombination method, 1) preparing a recombinant plasmid vector into which a DNA fragment encoding a lipase and a tliDEF ABC transporter derived from Pseudomonas sp. 2) preparing a Seratia Mason genus recombinant strain transformed with the Seratia Mason genus strain with the recombinant plasmid vector; 3) inducing the extracellular secretion of lipases by culturing the transformed Serratia genus recombinant strain in a medium; And 4) A lipase production method comprising the step of recovering the lipase from the culture medium of the transformed recombinant strain. The method of claim 9, The recombinant plasmid vector is a lipase production method, characterized in that it has a genetic map represented by any one of Figures 1a, 1b, 1c and 1d. The method of claim 9, Said Seratia genus genus strain is any one of SM2216 (KCTC2216), SM2356 (KCTC2356), SM2516 (KCTC2516), SM2798 (KCTC2798) and SM2801 (KCTC2801). The method of claim 9, Recombinant strains of the genus Serratia masons are strains of the original strain SM2356 (KCTC2356) and transformed with a recombinant plasmid vector derived from pKK223-3, strains of the original strain SM2798 (KCTC2798) and transformed with a recombinant plasmid vector derived from pKK223-3, Wherein the original strain is SM2356 (KCTC2356) and a strain transformed with a recombinant plasmid vector derived from pDSK519 and the original strain is SM2516 (KCTC2516) and a strain transformed with a recombinant plasmid vector derived from pDSK519 The lipase production method characterized in that.

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