KR20170021595A - Lipase with sn-1 specificity and method for production thereof - Google Patents

Abstract

The present invention relates to a lipase which has an amino acid sequence represented by sequence number 2, and which specifically hydrolyzes or esterifies only an sn-1 site of glycerin fatty acid ester. The present invention also relates to a production method thereof using Pichia yeast. According to the present invention, the use of the Cordyceps militaris-derived site-specific lipase targeting sn-1 enables the highly efficient production of biologically active substances and specific restructured lipids such as 1-monoglyceride and 2,3-diglyceride, as the site-specific lipase specifically acts on a number 1 site on a skeletal structure of glycerol.

Description

SN-1 POSITION SPECIFIC LIPHASE AND LIPASE WITH SN-1 SPECIFICITY AND METHOD FOR PRODUCTION THEREOF

The present invention having lipase sn -1 position specific lipase, and relates to its production method, and more particularly, to the amino acid sequence of SEQ ID NO: 2 or an ester hydrolysis to screen only the sn -1 position of glycerol fatty acid ester and a specific The present invention relates to a process for producing it using Pichia yeast.

Most of the enzymes currently in commercial use are found in nature, and 95% of all enzymes are derived from microorganisms. Enzymes discovered in the natural world may be used directly in actual industrial processes. However, recently, as the technology for designing and improving enzymes has developed, paradigms have been changed by optimizing enzymes and applying them to processes.

On the other hand, lipase used for new maintenance development such as reconstitution lipids is a recently attracted enzyme and is expected to activate the market in the future. It is expected that a new form of lipase with high activity and specificity will be secured and improved to excellent form by applying genetic manipulation and protein modification technology, leading to the market for high value added recombinant lipid production.

Lipase, which is widely distributed in nature from plants and animals to microorganisms, plays various roles such as bioconversion, emulsifier, flavor enhancement and specific organic synthesis. Therefore, lipase can be applied to food, pharmaceutical, cosmetics, Lt; / RTI >

Most lipases present in the natural world do not have a site specificity for substrates, which catalyze sn -1 or sn- 2 positions with sn- 1, 3 position-specific lipases catalyzing only positions 1 and 3 Specific lipases are rarely reported in the academia.

Enzyme-mediated Reconstitution In the production of lipids, enzyme activity and site-specific properties are among the most important factors. Currently, studies on the synthesis of reconstructed lipids which improve the physical, chemical and nutritional characteristics of lip using sn -1,3 position specific lipase are actively under way. Lipase having such characteristics is used for production of new functional material but it does not have complete position specificity by reacting sn- 1 and sn- 3, ie, positions 1 and 3 of glycerol skeleton together, There are still disadvantages.

On the other hand, the specific lipases sn -1 position having a high activity and specificity is located there and the functional lipid reorganization of monoglyceride (monoglyceride), di glyceride (diglyceride) can be synthesized in a high yield, high purity, the sn -1 position Due to its specific function, it can inhibit the formation of by-products during the synthesis of lipids by interesterification and thus can be considered as an enzyme with high added value. Furthermore, by analyzing the type of fatty acid at the sn- 1 position of triglyceride, it can be used as a breakthrough technique in the identification of lipase reaction mechanism.

Korean Patent Publication No. 10-2011-0121393 (Published Date: November 07, 2011) discloses a lipase produced from a strain of Pichia lynferdii NRRL Y-7723, and its use , This lipase is an enzyme secreted by the growth of Pichia rinferredi NRRL Y-7723 strain, and it is said that it maintains high activity even in the temperature range of 0 to 30 캜 at which the activity of the lipase enzyme is lowered.

In the present invention, a lipase enzyme capable of specifically hydrolyzing or esterifying only the sn- 1 position of the glycerol fatty acid ester is developed and provided.

The present invention provides a lipase having an amino acid sequence as set forth in SEQ ID NO: 2, which is capable of specifically hydrolyzing or esterifying the sn- 1 position.

In the present invention, Cordyceps militaris ) to obtain genetic information of novel lipases with high activity and sn- 1 locus specificity. PCR was used to amplify only the correct portion and inserted into the yeast vector to construct a vector for lipase expression. The constructed vector was introduced into a P. pastoris yeast strain, transformed, and then cultured to obtain a transformant. The sec -1 locus specific hydrolytic activity of lipase was finally confirmed by culturing the obtained transformants.

On the other hand, the present invention provides a method for producing lipase, wherein a strain of the genus Pichia is used as an expression host strain in producing lipase having the amino acid sequence of SEQ ID NO: 2. When the host strain Picaca is used, the lipase having the amino acid sequence shown in SEQ ID NO: 2 can be mass-produced in an active form. In this case, the Pichia spp. Strain may be, for example, Pichia pastoris .

On the other hand, in the lipase production method of the present invention, the lipase having the amino acid sequence of SEQ ID NO: 2 is a lipase produced by Cordyceps militaris . < / RTI > The lipase having the amino acid sequence shown in SEQ ID NO: 2 is a sn- 1 site-specific lipase.

On the other hand, the present invention provides a sn- 1 site-specific hydrolysis method characterized by using lipase having the amino acid sequence of SEQ ID NO: 2 in hydrolyzing the sn- 1 position of the glycerin fatty acid ester. Further, the present invention provides a sn- 1 position-specific esterification reaction method characterized by using a lipase having an amino acid sequence as set forth in SEQ ID NO: 2 in an esterification reaction in which a fatty acid is bonded to a glycerin sn- 1 position .

The lipase of the present invention can mediate both the hydrolysis and the esterification reaction. When the lipase of the present invention is used, the lipase specifically acts only on the 1-position of the glycerol backbone structure. Thus, the 1-monoglyceride and 2, Specific recombinant lipids such as 3-diglyceride and physiologically active substances can be produced with high efficiency.

The composition and positional distribution ( sn -1 (3), sn -2) of fatty acids located in the glycerol skeleton of the neutral lipid present in the natural world is changed chemically or enzymatically And the like, to improve the physical, chemical and nutritional characteristics of lipids.

Reconstituted lipids can be produced through chemical or enzymatic reactions.

The chemical reaction is carried out under high temperature and high pressure using a catalyst such as sodium methoxide, sodium / lithium hydroxide and the like, and thus the energy consumption is large. In addition, the byproducts are generated and are difficult to remove, and there is a disadvantage that complicated purification processes for removing harmful catalysts and removing them are complicated.

However, since the enzymatic reaction is carried out at a relatively mild temperature (30 to 60 ° C), side reactions do not occur and the purification process of the product is simpler than the chemical method. In addition, it exhibits substrate and position specificity and is suitable for customized maintenance and synthesis.

If the lipase enzyme of the present invention having the sn- 1 position specificity is used for the synthesis of trans-free, low-calorie, and functional reconstituted lipids, the production of byproducts due to the specific synthesis process can be suppressed, and a high yield and easy purification process .

That is, when the sn- 1 site-specific lyase derived from the Coryseps milliliterus of the present invention is used, it specifically acts only at the 1-position of the glycerol backbone structure, and thus, 1-monoglyceride and 2,3-diglyceride The present invention can be applied to the production of specific recombinant lipids and physiologically active substances with high efficiency.

The sn- 1 site-specific lipase derived from the Coryseps milliliterus of the present invention can also be used as an analytical method capable of distinguishing fatty acids located at sn- 1 and sn- 2 of triglyceride.

Figure 1 is a schematic diagram of Cordyceps < RTI ID = 0.0 > militaris. < / RTI >
Fig. 2 shows the reaction specificity ( sn- 1) of the lipase of the present invention.
Figure 3 shows the gene size of the lipase of the present invention.
4 is a nucleic acid sequence of the lipase gene of the present invention (SEQ ID NO: 1).
5 is a map of a recombinant vector for expression of the lipase of the present invention.
6 is a graph showing the expression of the lipase of the present invention through a recombinant vector.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following embodiments and experimental examples, and includes modifications of equivalent technical ideas.

[ Example  One: Cody's From Militaris  Lipase separation]

Cordyceps To separate and purify lipase from militaris , Coryseps milliliter extract was fractionated by ammonium sulfate precipitation method and applied to FPLC.

Prior to separation and purification of the enzyme using FPLC, the column was stabilized with 50 mM Tris-Cl buffer (pH 7.0), and the enzyme was separated according to the ionic strength using a HiTrap DEAE sepharose FF column (GE healthcare) Lt; / RTI > The functional group of this column is -N ⁺ (C ₂ H ₅ ) ₂ H, which forms a bond with the negative charge portion of the protein and separates the protein according to the degree of binding between protein and functional group, increasing the concentration of 1 M NaCl . The ionic strength unit was separated by 5% using step elution.

Proteins eluted through the column were identified as peaks by measuring the absorbance at 280 nm. Proteins eluted at 1.0 mL / min were fractionated into 5.0 mL. The fractions which showed the lipase activity at 5% NaCl concentration were obtained by collecting the fractionated proteins, and the pigment and unnecessary protein were removed and the purification degree was increased.

Partially separated proteins were collected and concentrated through anion exchange chromatography, and the enzymes were separated by hydrophobic strength using Hitrap Phenyl FF column (GE healthcare). When the salt concentration of the buffer is high, the hydrophobic part of the protein expressed mutually binds to the functional group of the column, and the protein is eluted by lowering the salt concentration.

After the column was stabilized with 50 mM Tris-Cl buffer at pH 7.0, the protein was separated by reducing the concentration of ammonium sulfate from 0% to 100% in 25% increments. The protein eluted at a rate of 0.7 mL / min was fractionated into 5.0 mL. The fractions were collected and concentrated, and the activity was measured. As a result, it was confirmed that the lipase activity exhibited the highest lipase activity at 75% hydrophobic concentration.

The partially separated proteins were collected by concentration according to the hydrophobic strength, and then purified using a Sephacryl S-100 column (GE healthcare). The fractions eluted at between 40 and 50 mL showed lipase activity at a flow rate of 0.4 mL / min using 50 mM Tris-Cl buffer containing 0.15 M NaCl.

The fractions showing lipase activity were pooled and concentrated, followed by repeated gel permeation chromatography. The separation conditions were the same as those of the gel permeation chromatography as described above, and it was confirmed that the separation showed a single peak. The purified lipase exhibited a yield of 2.47% and a purity of 94.54%, and was a monomer having a molecular weight of 60 kDa (FIG. 1).

[ Example  2: Example  Separated from 1 Cody's Millitaris  Identification of position specificity of derived lipase]

TLC was performed to confirm the position specificity of the lipase separated from the above.

The lipase enzyme isolated from the above-mentioned Coryseps milliliter was added to 50 mM 'Clark and Lubs' buffer (pH 9.0) supplemented with 20 mM triolein, and reacted at 40 ° C. for 40 hours. Then, 0.2 mL Of chloroform was added to separate the lipid layer.

Acetone / acetic acid (96: 4: 1, v / v / v) solvent was added to the silica gel plate (150 Å, Whatman) Lt; / RTI > Separated spots were developed using iodine vapor. As a result of TLC, it was confirmed that the main product of the enzymatic reaction was 1,2-diolein, specifically hydrolyzing only the 1-position of triolein (FIG. 2).

[ Example  3: Example  Separated from 1 Cody's Millis Derived phage  Analysis of molecular level characteristics]

MALDI-TOF analysis was used to obtain data on the lipase characterization and recombinant DNA technology that were isolated in Example 1.

After hydrolysis of the enzyme protein derived from Coryseps milliliterus with proteolytic enzymes, the mass of the cleaved peptide fragment (DLLWTNLLANR, VAAVIGDFVFTLAR, VGGFGFLAGSEVLEDGSTNLGLRDQR) was measured and compared with the protein information listed in the National Center for Biotechnology Information (NCBI) Respectively.

As a result, it showed high homology with the 1743 base pair of extracellular releasing lipase derived from Codishes milliliterus, which was confirmed by genome sequencing in China (Table 1).

Accession number ¹⁾ Precursor ( m / z ) Peptide (mass) Identified protein Database searched peptide MASCOT
SCORE G3J6X7 664.9 1327.8 Extracellular lipase, putative ⁵⁴⁷ DLLWTNLLANR ⁵⁵⁷ 62 G3J6X7 739.9 1477.8 Extracellular lipase, putative ⁴⁴⁹ VAAVIGDFVFFS ⁴⁶² 67 G3J6X7 898.8 2693.4 Extracellular lipase, putative ¹⁹² VGGFGFLAGSEVLEDGSTNLGLRDQR ²¹⁷ 71

1) Accession numbers from http://www.uniprot.org

Although the extracellular release lipase listed in NCBI has high homology with the partial amino acid sequence of the lipase isolated and purified by the present inventors, it has not been confirmed that it has the same DNA information as the Coryseps milliliter lipase used in the present invention.

Therefore, cDNA was prepared from the Coryseps milliliter nucleic acid possessed by the present inventors and subjected to DNA sequence analysis. The total RNA was extracted with Trizol reagent, which was pulverized using liquid nitrogen, and the cDNA was synthesized by reverse transcription using mRNA as a template. Subsequently, primers were prepared based on NCBI C-terminal and N-terminal upper gene and subgeneric base sequences, and cDNA was amplified using PCR as a template.

The size of the amplified PCR product was confirmed by agarose gel electrophoresis. As a result, amplification product was confirmed at a position larger than 1.5 kb and smaller than 2.0 kb (FIG. 3).

When DNA was extracted from the agarose gel and the nucleotide sequence was analyzed, it was found that 575 of the total 580 amino acids were identical (99%) when compared with the extracellular releasing lipase listed in NCBI at the amino acid level And 18 nucleotide mutations among 1740 nucleotides were confirmed (FIG. 4).

SEQ ID NO: 1 is the nucleic acid sequence of the lipase of the present invention, and SEQ ID NO: 2 is the amino acid sequence of the lipase of the present invention.

[ Example  4: Example  Separated from 1 Cody's Millitaris  Expression of Derived Lipase]

Based on the nucleotide sequence of Coryseps milliliter lipase, the following primers were prepared and subjected to PCR amplification using a primer.

CML_F: 5'-AGGGGTATCTCTCGAGAAAAGAGCGCCTCATTGCCCGGGA-3 '

CML_R: 5'-AGA AAGCTGGCGGCCGCGAAGTAGAGTACCTCCGTA-3 '

Each primer was artificially added to the Xho I / Not I recognition site so that it could be inserted into the Xho I and Not I sites of the pPICZ alpha -A vector. The PCR conditions were denaturation at 95 ° C for 3 minutes, followed by 30 cycles of PCR amplification. The denaturation was carried out at 95 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 120 seconds, and final extension at 72 ° C for 5 minutes in the last cycle Respectively. The DNA amplified by PCR was confirmed by determining agarose gel electrophoresis and gene base sequence.

The lipase gene amplified by PCR was subjected to Xho I / Not I restriction enzyme treatment, and the pPICZ alpha -A vector was similarly treated with the same enzyme Xho I / Not I restriction enzyme (FIG. 6). The ligation product was mixed with 100 μL of competent cells and then transformed by incubation at 42 ° C. for 1 minute.

The transformed Escherichia coli (E. coli) is adjusted to pH 7.5 and to a 'low salt LB medium (10 g tryptone, 5 g NaCl, 5 g yeast extract per 1L)' using a 10 N sodium hydroxide (NaOH) And Zeocin antibiotics (25 당 / mL) were added to each well.

After the plasmid DNA was extracted from the transformant, the recombinant plasmid pPICZa-CML vector capable of expressing the lipase was finally selected by confirming the DNA base sequence.

Using 10 μg of the purified pPICZα-CML DNA, the yeast Pichia to be used as a host ( Pichia pastoris) were transformed in KM71. First, the DNA was digested with Sac I restriction enzyme and made linear. The host strain, Pichia pastoris, was inoculated into 50 mL of YPD medium, incubated overnight at 30 ° C, and cultured until OD ₆₀₀ reached 0.9.

After culturing, the cells were centrifuged at 1,500 × g for 10 minutes, suspended in 25 mL of sterilized distilled water, centrifuged under the same conditions, and the same procedure was repeated with 100 mM LiCl to obtain a final volume of 0.4 mL . This compartment was mixed with linearized DNA, PEG, LiCl, and single-stranded DNA to a final volume of 0.4 mL and incubated at 30 ° C for 30 minutes. Then, it was immediately transformed by incubating at 42 DEG C for 25 minutes. YPD / Zeocin plate and cultured at 30 캜 until colonies were formed. In this step, the colonies formed in the above step were screened to determine whether they were transformed. The surviving transformants were selected by plating on YPD / Zeocin medium using the fact that the zeocin resistant gene contained in the vector is resistant to myosin present in the medium. Next, the transformed colonies were dissolved and PCR amplified to select transformants whose amplification products were confirmed.

On the other hand, of a lipase culture samples 420 μM p - to nitrophenyl palmitate (p -nitrophenyl palmitate) and placed in a 2.5 mL solution containing 0.2 M Tris-Cl buffer (pH 7.0) to be a 5 mL was reacted at 40 ℃ . After 15 minutes, 30 minutes, and 60 minutes of reaction, 500 μL of the reaction mixture was mixed with 0.1 M Tris-Cl buffer (pH 7.0), and a total of 1 mL of the mixture was added at 410 nm to the p -nitrophenyl palmitate decomposition product p- The absorbance of phenol was measured.

As a result, when the strain transformed with pPICZα-A vector was set as a control, significant increase in absorbance was observed only in the strain transformed with pPICZα-CML. These experimental results indicate that the lipase of the present invention was efficiently expressed in Pichia pastoris.

[ Example  5: Example  Separated from 1 Cody's Millitaris  Expression and Expression Characteristics of Derived Lipase by Host]

In this Example, the expression and expression characteristics of the lipase from Codisceps mililitaris isolated in Example 1 were compared by using different hosts. The host-specific expression vectors and strains were as shown in Table 2 below. Transformation methods and culturing conditions used a known method for the host and vector.

microbe Expression vector Host strain Expression and degree Soluble or insoluble expression E. coli















pET29-CML-C6His

BL21 (DE3) ++ I <I C43 (DE3) ++ I Origami (DE3) - - pET29-CML-MBP
BL21 (DE3) ++ I <I C41 (DE3) - pET26-CML-C6His

BL21 (DE3) ++ I C41 (DE3) + I C43 (DE3) + I pET26-CML-MBP
BL21 (DE3) + I <I C41 (DE3) + I <I pCold-CML-C6His


BL21 (DE3) - - C41 (DE3) - - C43 (DE3) - - Origami (DE3) + I pCold-CML-MBP

BL21 (DE3) ++ I <I C41 (DE3) + I <I Origami (DE3) - - Yeast pPICZα-CML Pichia pastoris KM71 ++ S Baculovirus pDualBac-CML Sf 9 + S

1) ++, high level of expression; +, low level of expression; -, no expression

2) S, soluble; I, insoluble

As a result of the experiment, the E. coli system was expressed as an insoluble inclusion body in which the lipase of the present invention was inactive. However, it was expressed as an active form soluble in baculovirus system such as Cordyceps. However, there was a problem that the expression amount of the expressed protein was small, and the baculovirus originally exhibited a small amount of expression and had a disadvantage that it was expensive to cultivate. However, the expression of the baculovirus as an active form means that the constructed cDNA sequence is correct.

On the other hand, when expressed in Pichia, it was confirmed to be expressed in a soluble form, and the production amount was higher than that of baculovirus.

As a result, it was confirmed that the Pichia strain was the most suitable host considering the culture cost and the expression level.

<110> Seoul National University R & DB Foundation <120> Lipase with sn-1 specificity and method for production thereof <130> AP-2015-0132 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1743 <212> DNA <213> Cordyceps militaris <400> 1 atgaaattct cacttgtggc tctggccact ctcccgtttt atgctgtcgc agcgcctcat 60 tgcccgggac aacccaatgg tccgcccagg gccgcggaag tgagggtcga tatacctggc 120 gccacaggcg gcacagtcat tggagtcgtc accgaagttg agagcttcaa cggcatcccc 180 tacgctgact gcccgtccgg tgcccttcga cttcgaccgc cgaggaagct ctcgcgcgcg 240 ctcggcgtct tcaacgccac ggccgccgcg ccggcctgtc cccagatgcc gcccaacacc 300 accgaagtgc tgctgccgcc gcttctcggc aaggacatga cgcccgagtt ctggccagac 360 gacctcatcc ggggccagga agactgcctc accgtcaacg tgcagcggcc caagggcacc 420 cgtgagggcg cccgcctgcc cgtgctcttc tacatctttg gaggcggctt cacggccggc 480 gccaccagcg ccaacaatgc cgaaaagttc ttgcggttcg ccgaggcgca gcagaagccg 540 cacccgttca tctttgtcgg cgtcaactac cgcgtcggcg ggttcgggtt cctcgccggc 600 agcgaggttc tcgaggacgg cagcaccaac ctcgggctcc gcgaccagcg gatgggcctc 660 ggtgggtgg cggacaacat tgcctacttt ggcggcgacc cggacagagt gaccatctgg 720 ggccagtcgt ccggctccat ctccgtgttt gaccagctcg cgctctacaa cggcaacgca 780 acctacaaac aaaagccgct tttccggagc gccatcatga actcgggcag cgtgattccc 840 acggagcggg tcgactcgca tcgggcgcag gccattttcg atgccgtcgt cgaagcggcc 900 aactgcagcg agccggccac ctccaagctg gactgtcttc gaaatgcctc ctttccgacc 960 ttctatcgcg cggcaaactc ggtcccgcgc atcttggaca actcgtctct cgccatctcc 1020 tacctgccgc ggcccgacgg ggagctgctg gcggacagtc ccgaggtcct tgccaacacg 1080 ggcaactact atgccgttcc cgccatcctc accaatcagg aggacgaagg aaccctgttc 1140 gctttcgccc agaggcacgt caacgacacc gacagcctcg tcgactatct caaggagacg 1200 tttttcgaca aggcgacaag ggagcaggtc gccggcctcg tggatacata cccagccgat 1260 tcggccgatg gaagcccgtt ccggactggt gatcagaacg agtggtacga ggaagcgtac 1320 ggtgccggca aaggcttcaa gagggtcgcg gctgtgatag gcgactttgt cttcacgctg 1380 gcccgtcgcc tggcgcttga tggcatggcc acttcgcatc cgacggtgcc tctttggtcc 1440 tctctgaact ccatggccca cgggattgtg ggcttctacg gtacgggaca cggtgcagat 1500 gtcaacatga tttttgaagg cattggcata cctgcgctga ctaccaggtc gtactatctg 1560 aatttcctat atactgccga cccgaataat ggtaccacgg aatttagaca gtggccaaag 1620 tggacgccac agggtaggga tttgctgtgg actaaccttt tggcgaatag agacttgaag 1680 gatactttca ggaatgatag ctatacattc ttgaaggaga atacggaggt actctacttc 1740 taa 1743 <210> 2 <211> 580 <212> PRT <213> Cordyceps militaris <400> 2 Met Lys Phe Ser Leu Ala Leu Ala Thr Leu Pro Phe Tyr Ala Val   1 5 10 15 Ala Ala Pro His Cys Pro Gly Gln Pro Asn Gly Pro Pro Arg Ala Ala              20 25 30 Glu Val Arg Asp Ile Pro Gly Ala Thr Gly Gly Thr Val Ile Gly          35 40 45 Val Val Thr Glu Val Glu Ser Phe Asn Gly Ile Pro Tyr Ala Asp Cys      50 55 60 Pro Ser Gly Ala Leu Arg Leu Arg Pro Pro Arg Lys Leu Ser Arg Ala  65 70 75 80 Leu Gly Val Phe Asn Ala Thr Ala Ala Ala Pro Ala Cys Pro Gln Met                  85 90 95 Pro Pro Asn Thr Thr Glu Val Leu Leu Pro Pro Leu Leu Gly Lys Asp             100 105 110 Met Thr Pro Glu Phe Trp Pro Asp Asp Leu Ile Arg Gly Gln Glu Asp         115 120 125 Cys Leu Thr Val Asn Val Gln Arg Pro Lys Gly Thr Arg Glu Gly Ala     130 135 140 Arg Leu Pro Val Leu Phe Tyr Ile Phe Gly Gly Gly Phe Thr Ala Gly 145 150 155 160 Ala Thr Ser Ala Asn Ala Glu Lys Phe Leu Arg Phe Ala Glu Ala                 165 170 175 Gln Gln Lys Pro His Phe Ile Phe Val Gly Val Asn Tyr Arg Val             180 185 190 Gly Gly Phe Gly Phe Leu Ala Gly Ser Glu Val Leu Glu Asp Gly Ser         195 200 205 Thr Asn Leu Gly Leu Arg Asp Gln Arg Met Gly Leu Glu Trp Val Ala     210 215 220 Asp Asn Ile Ala Tyr Phe Gly Asp Pro Asp Arg Val Thr Ile Trp 225 230 235 240 Gly Gln Ser Ser Gly Ser Ile Ser Val Phe Asp Gln Leu Ala Leu Tyr                 245 250 255 Asn Gly Asn Ala Thr Tyr Lys Gln Lys Pro Leu Phe Arg Ser Ala Ile             260 265 270 Met Asn Ser Gly Ser Val Ile Pro Thr Glu Arg Val Asp Ser His Arg         275 280 285 Ala Gln Ala Ile Phe Asp Ala Val Val Glu Ala Ala Asn Cys Ser Glu     290 295 300 Pro Ala Thr Ser Lys Leu Asp Cys Leu Arg Asn Ala Ser Phe Pro Thr 305 310 315 320 Phe Tyr Arg Ala Asn Ser Val Pro Arg Ile Leu Asp Asn Ser Ser                 325 330 335 Leu Ala Ile Ser Tyr Leu Pro Arg Pro Asp Gly Glu Leu Leu Ala Asp             340 345 350 Ser Pro Glu Val Leu Ala Asn Thr Gly Asn Tyr Tyr Ala Val Pro Ala         355 360 365 Ile Leu Thr Asn Gln Glu Asp Glu Gly Thr Leu Phe Ala Phe Ala Gln     370 375 380 Arg His Val Asn Asp Thr Asp Ser Leu Val Asp Tyr Leu Lys Glu Thr 385 390 395 400 Phe Phe Asp Lys Ala Thr Arg Glu Gln Val Ala Gly Leu Val Asp Thr                 405 410 415 Tyr Pro Ala Asp Ser Ala Asp Gly Ser Pro Phe Arg Thr Gly Asp Gln             420 425 430 Asn Glu Trp Tyr Glu Glu Ala Tyr Gly Ala Gly Lys Gly Phe Lys Arg         435 440 445 Val Ala Val Ile Gly Asp Phe Val Phe Thr Leu Ala Arg Arg Leu     450 455 460 Ala Leu Asp Gly Met Ala Thr Ser His Pro Thr Val Pro Leu Trp Ser 465 470 475 480 Ser Leu Asn Ser Met Ala His Gly Ile Val Gly Phe Tyr Gly Thr Gly                 485 490 495 His Gly Ala Asp Val Asn Met Ile Phe Glu Gly Ile Gly Ile Pro Ala             500 505 510 Leu Thr Thr Arg Ser Tyr Tyr Leu Asn Phe Leu Tyr Thr Ala Asp Pro         515 520 525 Asn Asn Gly Thr Thr Glu Phe Arg Gln Trp Pro Lys Trp Thr Pro Gln     530 535 540 Gly Arg Asp Leu Leu Trp Thr Asn Leu Leu Ala Asn Arg Asp Leu Lys 545 550 555 560 Asp Thr Phe Arg Asn Asp Ser Tyr Thr Phe Leu Lys Glu Asn Thr Glu                 565 570 575 Val Leu Tyr Phe             580

Claims (7)

a lipase having an amino acid sequence as set forth in SEQ ID NO: 2, which is capable of specifically hydrolyzing or esterifying the sn- 1 position.
In producing the lipase having the amino acid sequence shown in SEQ ID NO: 2,
Wherein the host strain for expression is a strain of the genus Pichia .
3. The method of claim 2,
The above-
Pichia &lt; / RTI &gt; pastoris ).
3. The method of claim 2,
The lipase having the amino acid sequence of SEQ ID NO:
Cordyceps militaris. &lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
3. The method of claim 2,
The lipase having the amino acid sequence of SEQ ID NO:
sec -1 position-specific lipase.
According, sn -1 position-specific hydrolysis methods characterized by using a lipase having the amino acid sequence shown in SEQ ID NO: 2 as the hydrolysis of the sn -1 position of glycerol fatty acid ester.
A sn- 1 site-specific esterification reaction method characterized by using a lipase having an amino acid sequence as set forth in SEQ ID NO: 2 in an esterification reaction in which a fatty acid is bonded to the sn- 1 position of glycerin.

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