KR102065239B1 - Low temperature activated lipase and producing method thereof - Google Patents

Abstract

The present invention relates to a low temperature active lipase and a method for producing the same, and since the lipase is a novel lipase that maintains high activity at low temperature and is easily inactivated by temperature, the lipase is effectively used in pharmaceuticals, foods, detergents, cosmetics, and biodiesel. It can be used in various industrial fields such as being used as a catalyst in production.

Description

Low temperature activated lipase and production method thereof

The present invention relates to a low temperature active lipase and a method for producing the same, and more particularly, derived from xantinobacterium PAMC25641 isolated from a microorganism inhabiting the polar region, maintaining high activity at low temperature and easy inactivation by temperature. One novel lipase relates.

Lipase (lipase, triacylglycerol acylhydrolase EC 3.1.1.3) is a glycerol ester hydrolase, an enzyme that hydrolyzes triacylglycerol to free fatty acids and glycerol.

Lipases promote not only the hydrolytic activity of triglycerides but also esterification, transesterification reactions, acid degradation, alcohol degradation and amino degradation. Lipases usually exhibit high chemical selectivity, regioselectivity and enantioselectivity, and also have high substrate specificity. The properties of these lipases can be usefully used in various industries, such as those used as catalysts in pharmaceutical, food, detergent, cosmetic, and biodiesel production.

In addition, industrially used lipases are mainly produced by microorganisms such as fungi and bacteria. Lipases derived from microorganisms are very useful for industrial use because enzyme properties vary widely depending on their origin and the type of lipase produced. .

In particular, low temperature lipases isolated from microorganisms growing in low temperature environments such as polar regions exhibit high catalytic activity at low temperatures compared to other mesophilic and thermophilic lipases. Therefore, low temperature lipase can be very useful in food, pharmaceutical industry, etc., where the reaction should be performed at a relatively low temperature.

However, there is a problem that it is difficult to economically mass-produce an enzyme required for industrialization using this strain directly. Therefore, it is necessary to develop a technique for isolating and identifying a lipase gene from this strain and mass-producing it in another expression host.

Therefore, the present inventors cultured Escherichia coli transformed with a nucleic acid sequence encoding the amino acid sequence represented by SEQ ID NO: 1 derived from Xantinobacterium PAMC25641, the recombinant lipase purified from the cell extract supernatant of E. coli has high stability at low temperature. And activity was confirmed.

Accordingly, it is an object of the present invention to provide a lipase comprising the amino acid sequence represented by SEQ ID NO: 1.

Another object of the present invention is to provide a recombinant vector comprising a nucleic acid sequence encoding the amino acid sequence represented by SEQ ID NO: 1.

Still another object of the present invention is to provide a microorganism transformed with a vector comprising a nucleic acid sequence encoding the amino acid sequence represented by SEQ ID NO: 1.

Another object of the present invention is to provide a lipase production method comprising the following steps:

Transforming the microorganism with a vector comprising a nucleic acid sequence encoding an amino acid sequence represented by SEQ ID NO: 1;

Microbial culture step of culturing the transformed microorganism; And

Protein purification step of separating the lipase from the microbial culture.

The present invention relates to a low temperature active lipase and a method for producing the same, wherein the lipase according to the present invention is derived from xantinobacterium PAMC25641 isolated from a microorganism in the polar region, and maintains high activity at low temperature and is inactivated by temperature. Indicates ease of use.

The present inventors confirmed that when culturing Escherichia coli transformed with the nucleic acid sequence represented by SEQ ID NO: 2 derived from Xanthinobacterium PAMC25641, the recombinant lipase purified from the cell extract supernatant of E. coli shows high stability and activity at low temperature. It was.

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

One aspect of the invention is a lipase comprising the amino acid sequence represented by SEQ ID NO: 1.

The lipase may be encoded by a nucleic acid sequence represented by SEQ ID NO: 2, but is not limited thereto.

The lipase may be derived from a strain of genus Xanthinobacterium, for example, but may be derived from xanthinobacterium PAMC25641, but is not limited thereto.

The lipase may have a hydrolytic activity of 60% or more at 5 to 15 ° C.

In one embodiment of the invention, the lipase comprising the amino acid sequence represented by SEQ ID NO: 1 showed the highest activity at pH 7.0 conditions of 35 ℃, 70% at 15 ℃, 60% at 10 ℃ . Stability was maintained at 80% or higher at 5 to 15 ° C., but activity was rapidly decreased at 20 ° C. or higher, and thus it may be regarded as a low temperature lipase.

The lipase may be one that selectively hydrolyzes an acyl group having 4 to 14 carbon atoms, an acyl group having 4 to 12 carbon atoms, or an acyl group having 4 to 10 carbon atoms, for example, an acyl group having 8 carbon atoms, but is not limited thereto.

Another aspect of the invention is a vector comprising a nucleic acid sequence encoding the amino acid sequence represented by SEQ ID NO: 1.

As used herein, the term "vector" means a means for expressing a gene of interest in a host cell. For example, viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors are included. Vectors that can be used as the recombinant vector are plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14). , pGEX series, pET series and pUC19, etc.), phage (e.g., λgt4λB, λ-Charon, λΔz1 and M13, etc.) or viruses (e.g., SV40, etc.) For example, it may be a pET-28a (+) vector, but is not limited thereto.

The recombinant vector can typically be constructed as a vector for cloning or a vector for expression. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals or microorganisms. The recombinant vector may be constructed through various methods known in the art.

The recombinant vector may be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector used is an expression vector and the prokaryotic cell is a host, a strong promoter capable of promoting transcription (for example, a pL ^λ promoter, a CMV promoter, a trp promoter, a lac promoter, a tac promoter, T7 promoters, etc.), ribosome binding sites for initiation of translation, and transcription / detox termination sequences. In the case of eukaryotic cells as hosts, replication origins that operate in eukaryotic cells included in the vector include f1 origin, SV40 origin, pMB1 origin, adeno origin, AAV origin, and BBV origin. It is not limited. In addition, promoters derived from the genome of mammalian cells (eg, metallothionine promoters) or promoters derived from mammalian viruses (eg, adenovirus late promoters, vaccinia virus 7.5K promoters, SV40 promoters, Cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

In one embodiment of the present invention, a transformant may be made by inserting a recombinant vector into a host cell, and the transformant may be obtained by introducing the recombinant vector into an appropriate host cell.

The host cell may be any host cell known in the art as a cell capable of continuously cloning or expressing the expression vector while being stable.

Host cells used in the present invention may include E. coli, yeast, animal cells, plant cells, insect cells and the like, prokaryotic cells, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus genus strains, such as Bacillus thuringiensis, and Salmonella typhimurium, Serratia marsonsons and Enterobacteria and strains such as various Pseudomonas species, and when transforming eukaryotic cells as host cells, yeast (Saccharomyce cerevisiae), insect cells, plant cells and animal cells, such as Sp2 / 0, CHO (Chinese) hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell line, etc. may be used, but is not limited thereto.

The transport (introduction) of the polynucleotide or the recombinant vector including the same into the host cell may employ a transport method well known in the art. For example, when the host cell is a prokaryotic cell, a CaCl ₂ method or an electroporation method may be used. When the host cell is a eukaryotic cell, a micro-injection method, calcium phosphate precipitation method, an electroporation method, Liposome-mediated transfection and gene balm and the like can be used, but are not limited thereto.

The method of selecting the transformed host cell can be easily carried out according to methods well known in the art using a phenotype expressed by a selection label. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

The nucleic acid sequence may be derived from a strain belonging to the genus Xanthinobacterium, for example, may be derived from Xanthinobacterium PAMC25641, but is not limited thereto.

Another aspect of the invention is a microorganism transformed with a vector comprising a nucleic acid sequence encoding the amino acid sequence represented by SEQ ID NO: 1.

The nucleic acid sequence may be derived from a strain belonging to the genus Xanthinobacterium, for example, may be derived from Xanthinobacterium PAMC25641, but is not limited thereto.

The microorganism is composed of Escherichia coli, Spirulina platensis, Chlamydomonas reinhardtii, Haematococcus pluvialis and Chlorella vulgaris. It may be selected.

Another aspect of the invention is a lipase production method comprising the following steps:

Transforming the microorganism with a vector comprising a nucleic acid sequence encoding an amino acid sequence represented by SEQ ID NO: 1;

Microbial culture step of culturing the transformed microorganism; And

Protein purification step of separating the lipase from the microbial culture.

The amino acid sequence represented by SEQ ID NO: 1 may be derived from a strain of the genus Xantinobacterium, for example, may be derived from Xantinobacterium PAMC25641, but is not limited thereto.

The lipase may have a hydrolytic activity of 60% or more at 5 to 15 ° C.

The lipase may be one that selectively hydrolyzes an acyl group having 4 to 14 carbon atoms.

The microorganism may be selected from the group consisting of Escherichia coli, Spirulina pratensis, Chlamydomonas Reinhardty, Hamatococcus prubilis and Chlorella vulgaris.

The present invention relates to a low temperature active lipase and a method for producing the same, and since the lipase is a novel lipase that maintains high activity at low temperature and is easily inactivated by temperature, the lipase is effectively used in pharmaceuticals, foods, detergents, cosmetics, and biodiesel. It can be used in various industrial fields such as being used as a catalyst in production.

1 is a schematic diagram of a recombinant vector inserted with a nucleic acid sequence encoding a lipase.
Figure 2 shows the phylogenetic tree of xantinobacterium species PAMC25617.
Figure 3a is a photograph showing the recombinant lipase SDS-PAGE according to an embodiment of the present invention.
Figure 3b is a photograph showing the SDS-PAGE purified by a column of the recombinant lipase Ni ^{2 +} -NTA affinity resin according to an embodiment of the present invention.
Figure 4a is a graph showing the change in activity of recombinant lipase with temperature conditions.
Figure 4b is a graph showing the change in stability of recombinant lipase with temperature conditions.
Figure 4c is a graph showing the change in activity of recombinant lipase according to pH conditions.
Figure 4d is a graph showing the stability change of recombinant lipase according to the pH conditions.
5 is a graph showing substrate specificity of recombinant lipase.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1 Isolation of Lipase Gene

Xanthinobacterium sp. PAMC25641 was distributed and cultured through the Polar and Alpine Microbial Collection, Korea Polar Research Institute, Incheon, and lipase gene was isolated by genomic DNA analysis. 50 ml liquid medium (0.5 g / L yeast extract, 0.5 g / L proteose peptone NO.3, 0.5 g / L casamino acids, 0.5 g / L C ₆ H ₁₂ O ₆ 0.5 g / L, soluble for culturing xantinobacterium PAMC25641) starch 0.5 g / L, C ₃ H ₃ NaO ₃ 0.3 g / L, HK ₂ O ₄ P 0.3 g / L, MgSO ₄ · 7H ₂ O 0.05 g / L, pH 7.2 in composition) in agar (BD, Franklin Lakes, NJ, USA) plates were inoculated with single colonies and incubated at 16 ° C. for 3 days.

Genomic DNA was isolated from the isolated strain using a genomic DNA mini kit (genomic DNA mini kit; Invitrogen, Carlsbad, CA, USA). Analysis of the isolated genomic DNA revealed a protein consisting of 309 amino acids (SEQ ID NO: 1) and a predicted molecular weight of 32700 Da, and a 930 bp long nucleic acid sequence (SEQ ID NO: 2) encoding the same.

SEQ ID NO: designation order One Lipase from Xantinobacterium PAMC25641 MSLDPLIAAWLEESKNAPPPASLADLRAATETGLRQLHGPMEDVASIRDYVLPGEHALIVRAYVPAGADTSSPLPAIVFAHGGGWCLCSLDLYDNPCRALANATGCVIFSVDYRLAPEHKFPVPLADFYRALCWVAQHAAVLGIDAKRLAVGGDSAGGNLAAAAALMARDQAGPALAHQLLLYPALDFAFDTASYQRYAEGHSLTREAMRFCWSAYLNAPAEGSNPYAAPLRAASLKDLPPATVLVCEFDPLQDEGAAYARRLRDDGVAAQCVQLDGMIHASIHMPGLTPAARGLFDVAGLAMRKALGR 2 Nucleic acid sequence encoding lipase derived from xanthinobacterium PAMC25641 atgagcctcgatccactgatcgccgcctggctggaagagagcaaaaatgcgccgccgcccgcctcgctggcagacctgcgcgccgccacggagacgggcttgcgccagctgcacggaccgatggaggacgtcgcctcgatcagggattacgtgctgccgggcgaacatgcgctcatcgtgcgcgcctacgtgccggctggcgcggatacgagttcccccctgcccgccatcgtctttgcgcatggcggcggatggtgcctgtgctcgctggacctgtacgacaatccgtgccgcgcgctggccaacgccaccggctgcgtgatcttttccgtcgactaccggctggcgcccgagcacaagttccccgtgccgctagccgacttttaccgggcactatgctgggtcgcccagcatgccgcagtgctgggcatcgacgcgaagcgcctcgccgtgggcggcgacagcgcgggcggcaacctggccgccgcagcggcactgatggcgcgcgaccaggccggtcccgcgctggcgcaccagttgctgctgtacccggcgctcgatttcgccttcgacacggcttcgtaccagcgctatgcggagggccattcgctgacgcgggaagcgatgcgtttttgctggtcggcctacctgaacgcgccggcagaagggagcaatccgtatgcggcaccgctgcgggccgcctcactgaaggacttgccaccggcgacggtgctggtgtgcgaattcgatccgctgcaggacgagggcgcggcgtatgcgcggcgcttgcgcgatgacggcgtggcggcgcaatgcgtgcagctcgacggcatgatccatgcatcgatacacatgccgggcctgacgccagcggcgcggggattgtttgacgtggcgggactggcgatgcggaaggcgctgggaaggtaa

Example 2: Lipase Gene Cloning

In order to clone the gene of the lipase isolated in Example 1, primers of SEQ ID NOs: 3 and 4 were used as shown in Table 2 below.

SEQ ID NO: designation Sequence (5 '-> 3') 3 PAMC25641_Lip-F CCC GGATCC ATGAGCCTCGATCCACTGAT 4 PAMC25641_Lip-R CCC AAGCTT TTACCTTCCCAGCGCCT

Forward primer's GGA TCC 'is Bam I restriction enzyme (NEB, Ipswitch, MA, USA) is a cleavage site for recognition, from the reverse primer' AAG CTT 'is a cleavage site recognized by Hind III restriction enzymes (Promega, Fitchburg, Wis, USA). Gene amplification was carried out using a known polymerase chain reaction (PCR) using a set of primers prepared.

In order to ligation the amplified gene to pET28a (+) (Invitrogen, Carlsbad, CA, USA), the plasmid and amplified lipase genes were digested with BamHI and HindIII restriction enzymes, respectively, and then treated with ligase. . This recombinant plasmid was named pET28a (+): PAMC25641_lipase and is shown in FIG. 1.

E. coli (DH5α) strain (Invitrogen, Carlsbad, CA, USA), a transformation host, was transformed using electroporation. Transformed Escherichia coli (DH5α) was selected in Luria Bertani (LB; BD, Franklin Lakes, NJ, New Jersey, USA) medium containing 50 ug / ml kanamycin antibiotic, and the recombinant plasmid was plasmid mini Purified and sequenced with a plasmid mini kit (Invitrogen, Carlsbad, Calif., USA).

Example 3: Sequencing Analysis of Recombinant Lipase

Using the results of sequencing in Example 2, NCBI's BLAST program was used to confirm the identity of the lipase sequence. The nucleotide sequence was translated into an amino acid sequence using the SPA (Swiss Institute of Bioinformatics) ExPASy (Expert Protein Analysis System) translation tool (Translation tool).

As can be seen in Figure 2, the amino acid sequence is clustered (clustering) lipase protein of 15 highly homologous strains using the BLASTP program of NCBI through the phylogenetic analysis, most of the xantino other than the xantinobacterium PAMC25641 lipase It was confirmed that it is associated with bacterium lipase.

Example 4 Expression of Lipase from a Transformant

Escherichia coli as host cell for heterologous expression of lipase coli , E. coli ) BL21 (DE) (Invitrogen, Carlsbad, Calif., USA) was prepared and plasmid pET-28a (+) (Invitrogen, Carlsbad, Calif., USA) was used as a control vector.

The transformed E. coli was named BL21 (DE3). Luria Bertani (LB; BD, Franklin Lakes, NJ, New Jersey, USA) medium containing 50 ug / ml of kanamycin (Kanamycin; Thermo scientific, Waltham, Mass, USA) was maintained at 37 ° C, The transformed E. coli was added and cultured. When the OD ₆₀₀ of E. coli is 0.3 to 0.5, 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG; Bio basic, Markham, Ont, Canada) is used as a protease. Lipase expression was induced by addition and incubation at 16 ° C. for 15 hours 30 minutes.

Example 5: Isolation and Purification of Expressed Lipase

Recombinant lipase expressed according to Example 4 was analyzed via SDS-PAGE. Lipase activity was confirmed in the recombinant lipase expressed in the cell extract supernatant, because the expressed recombinant lipase comprises a His ₆ -tag Ni ^{2 +} -NTA column of affinity resin to increase the activity (Elpis biotiech, Dea-jeon, South Lipase was purified through Korea.

Specifically, after expression through IPTG induction, the cell culture was centrifuged at 4 ° C., 4,000Хg for 20 minutes to recover the inferior liquor, and 0.05 M tris-hydrochloric acid for separation of lipase protein formed in a native state. (Tris-HCl) (pH 8.0), 0.1 M sodium chloride (Nacl), 10% (v / v) glycerol (0.1 mg / ml) and lysozyme (0.1 mg / mL) was treated for 1 hour 30 at 16 ℃ After the reaction for minutes, the cells were disrupted by sonication, and the supernatant was recovered by centrifugation at 4 ° C and 13000 rpm for 10 minutes.

As shown in Figure 3a, it was confirmed in lane3 and lane7 overexpression of recombinant lipase in the cell extract supernatant expressed at 16 ℃ (M: protein size marker, lane1: pET28a (+): PAMC25641_lipase not induced expression) Cell extract, lane2: pET28a (+) cell extract that did not induce expression, lane3: expressed pET28a (+): PAMC25641_lipase cell extract, lane4: expressed pET28a (+) cell extract, lane5: pET28a that did not induce expression +): PAMC25641_lipase cell extraction supernatant, lane6: pET28a (+) cell extraction supernatant that did not induce expression, lane7: expressed pET28a (+): PAMC25641_lipase cell extraction supernatant, lane8: expressed pET28a (+) cell extraction supernatant).

Recovering the supernatant was washed with the Ni ^{2 +} -NTA a column of affinity resin was reacted for 1 hour 30 minutes 0.05 M sodium phosphate, J. (pH 8.0), 0.5 M sodium chloride (NaCl) and 0.02 M imidazole (Imidazole) Lipase was eluted at a volume of 6 ml. The concentration of eluted protein was measured by Bradford (Bradford M, Anal Biochem , 72, 248-254, 1976).

Subsequently, 0.05 M Tris-HCl (pH 8.0), 0.5 M Sodium Chloride (NaCl), 10% (v / v) Glycerol to measure the activity of recombinant lipase and to inhibit disulfide bonds between proteins And dialysis with 0.005 M 2-mercaptoethanol.

As can be seen in Figure 3b, the separation was able to observe the band at the position of 35.0 kDa greater than 32.7 kDa, it was confirmed that corresponds to the size estimated from the amino acid sequence containing His ₆ -tag (M: protein size marker , lane1: lipase cell extraction supernatant, lane2: non-column bound protein, lane3,4,5: wash with 20 mM imidazole buffer, lane6,7,8: elution with 350 mM imidazole.

Experimental Example  1: Confirmation of activity and stability according to temperature and pH of purified lipase

In order to confirm the biochemical properties of the purified recombinant lipase, colorimetric anaylsis was performed using p-NP octanoate as a substrate in the temperature range of 5 to 70 ° C and pH 4.0 to 9.0. Was performed.

As can be seen from Tables 3 and 4 and FIGS. 4A and 4B, the recombinant lipase showed the highest activity at 35 ° C. In addition, the temperature stability of the recombinant lipase was the most stable at 5 to 15 ℃, sharply decreased above 15 ℃.

Temperature (℃) 5 10 15 20 25 35 50 60 70 Relative activity (%) 55.76 60.43 68.59 87.65 93.16 100 49.69 46.57 39.21

Temperature (℃) 5 10 15 20 25 35 50 60 70 Stability 1h (%) 94.43 95.71 96.89 96.77 92.31 61.49 2.32 1.67 1.50 Stability 18h (%) 85.34 86.53 86.56 58.88 32.67 3.05 1.63 1.12 1.20

As can be seen from Tables 5 and 6 and FIGS. 4C and 4D, recombinant lipases exhibited the highest activity at pH 7.0. In addition, the pH stability of the recombinant lipase maintained at least 60% activity at pH 6.0 to 7.0.

pH 3 4 5 6 7 8 9 Relative activity (%) 6.27 16.98 21.93 26.51 100.00 95.56 75.26

pH 3 4 5 6 7 8 9 Stability 1h (%) 61.75 70.00 99.23 96.66 90.15 69.64 28.43 Stability 18h (%) 9.67 30.00 53.24 80.88 71.37 29.34 4.85

Therefore, it was confirmed that the recombinant lipase of the present invention is a cold-adapted lipase because it maintains high stability at low temperatures.

Considering that most low temperature lipases are known to maintain high activity at 5 to 25 ° C, the recombinant lipase of the present invention is rapidly reduced in activity at 15 ° C or higher, and therefore, it is expected to be useful in post-treatment processes.

Experimental Example  2: Confirmation of Substrate Specificity of Recombinant Lipase

In order to confirm the substrate specificity of the purified recombinant lipase, the hydrolytic activity of p-nitrophenyl ester having various chain lengths was confirmed. The p-nitrophenyl esters include pNP-butyrate (pNP-butyrate (C4), pNP-octanoate (C8), pNP-decanoate (C10), pNP-dodecanoate (pNP-dodecanoate, C12) and pNP-myristate (pNP-myristate, C14) were used. Hydrolytic activity was determined by colorimetric analysis.

As can be seen in Table 7 and FIG. 5, recombinant lipase showed the highest activity when pNP-octanoate (C8) was used as a substrate, and relatively low activity in the long carbon chain of C10 to C14.

temperament C4 C8 C10 C12 C14 Relative activity (%) 86.30 100.00 13.34 6.87 2.72

<110> Industry-University Cooperation Foundation Chonnam University <120> Low temperature activated lipase and producing method <130> PN170381 <160> 4 <170> KopatentIn 2.0 <210> 1 <211> 309 <212> PRT <213> Janthinobacterium PAMC25641 <400> 1 Met Ser Leu Asp Pro Leu Ile Ala Ala Trp Leu Glu Glu Ser Lys Asn   1 5 10 15 Ala Pro Pro Pro Ala Ser Leu Ala Asp Leu Arg Ala Ala Thr Glu Thr              20 25 30 Gly Leu Arg Gln Leu His Gly Pro Met Glu Asp Val Ala Ser Ile Arg          35 40 45 Asp Tyr Val Leu Pro Gly Glu His Ala Leu Ile Val Arg Ala Tyr Val      50 55 60 Pro Ala Gly Ala Asp Thr Ser Ser Pro Leu Pro Ala Ile Val Phe Ala  65 70 75 80 His Gly Gly Gly Trp Cys Leu Cys Ser Leu Asp Leu Tyr Asp Asn Pro                  85 90 95 Cys Arg Ala Leu Ala Asn Ala Thr Gly Cys Val Ile Phe Ser Val Asp             100 105 110 Tyr Arg Leu Ala Pro Glu His Lys Phe Pro Val Pro Leu Ala Asp Phe         115 120 125 Tyr Arg Ala Leu Cys Trp Val Ala Gln His Ala Ala Val Leu Gly Ile     130 135 140 Asp Ala Lys Arg Leu Ala Val Gly Gly Asp Ser Ala Gly Gly Asn Leu 145 150 155 160 Ala Ala Ala Ala Ala Leu Met Ala Arg Asp Gln Ala Gly Pro Ala Leu                 165 170 175 Ala His Gln Leu Leu Leu Tyr Pro Ala Leu Asp Phe Ala Phe Asp Thr             180 185 190 Ala Ser Tyr Gln Arg Tyr Ala Glu Gly His Ser Leu Thr Arg Glu Ala         195 200 205 Met Arg Phe Cys Trp Ser Ala Tyr Leu Asn Ala Pro Ala Glu Gly Ser     210 215 220 Asn Pro Tyr Ala Ala Pro Leu Arg Ala Ala Ser Leu Lys Asp Leu Pro 225 230 235 240 Pro Ala Thr Val Leu Val Cys Glu Phe Asp Pro Leu Gln Asp Glu Gly                 245 250 255 Ala Ala Tyr Ala Arg Arg Leu Arg Asp Asp Gly Val Ala Ala Gln Cys             260 265 270 Val Gln Leu Asp Gly Met Ile His Ala Ser Ile His Met Pro Gly Leu         275 280 285 Thr Pro Ala Ala Arg Gly Leu Phe Asp Val Ala Gly Leu Ala Met Arg     290 295 300 Lys Ala Leu Gly Arg 305 <210> 2 <211> 930 <212> DNA <213> Janthinobacterium PAMC25641 <400> 2 atgagcctcg atccactgat cgccgcctgg ctggaagaga gcaaaaatgc gccgccgccc 60 gcctcgctgg cagacctgcg cgccgccacg gagacgggct tgcgccagct gcacggaccg 120 atggaggacg tcgcctcgat cagggattac gtgctgccgg gcgaacatgc gctcatcgtg 180 cgcgcctacg tgccggctgg cgcggatacg agttcccccc tgcccgccat cgtctttgcg 240 catggcggcg gatggtgcct gtgctcgctg gacctgtacg acaatccgtg ccgcgcgctg 300 gccaacgcca ccggctgcgt gatcttttcc gtcgactacc ggctggcgcc cgagcacaag 360 ttccccgtgc cgctagccga cttttaccgg gcactatgct gggtcgccca gcatgccgca 420 gtgctgggca tcgacgcgaa gcgcctcgcc gtgggcggcg acagcgcggg cggcaacctg 480 gccgccgcag cggcactgat ggcgcgcgac caggccggtc ccgcgctggc gcaccagttg 540 ctgctgtacc cggcgctcga tttcgccttc gacacggctt cgtaccagcg ctatgcggag 600 ggccattcgc tgacgcggga agcgatgcgt ttttgctggt cggcctacct gaacgcgccg 660 gcagaaggga gcaatccgta tgcggcaccg ctgcgggccg cctcactgaa ggacttgcca 720 ccggcgacgg tgctggtgtg cgaattcgat ccgctgcagg acgagggcgc ggcgtatgcg 780 cggcgcttgc gcgatgacgg cgtggcggcg caatgcgtgc agctcgacgg catgatccat 840 gcatcgatac acatgccggg cctgacgcca gcggcgcggg gattgtttga cgtggcggga 900 ctggcgatgc ggaaggcgct gggaaggtaa 930 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 3 cccggatcca tgagcctcga tccactgat 29 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Artificial sequences <400> 4 cccaagcttt taccttccca gcgcct 26

Claims (15)

Lipase consisting of the amino acid sequence represented by SEQ ID NO: 1. The lipase of claim 1, wherein the lipase is encoded by a nucleic acid sequence represented by SEQ ID NO: 2. The lipase of claim 1, wherein the lipase is derived from a strain of genus Jantinobacterium. The lipase of claim 1, wherein the lipase has a hydrolytic activity of 60% or more at 5 to 15 ° C. The lipase of claim 1, wherein the lipase selectively hydrolyzes an acyl group having 4 to 14 carbon atoms. A recombinant vector comprising a nucleic acid sequence encoding the amino acid sequence represented by SEQ ID NO: 1. The recombinant vector of claim 6, wherein the nucleic acid sequence is derived from Xantinobacterium PAMC25641. Escherichia coli transformed with a vector comprising a nucleic acid sequence encoding the amino acid sequence represented by SEQ ID NO: 1. The Escherichia coli according to claim 8, wherein the nucleic acid sequence is derived from Xantinobacterium PAMC25641. delete Lipase production method comprising the following steps:
Transforming Escherichia coli with a vector comprising a nucleic acid sequence encoding the amino acid sequence represented by SEQ ID NO: 1;
Microbial culture step of culturing the transformed Escherichia coli; And
Protein purification step of separating lipase from E. coli culture. The method of claim 11, wherein the amino acid sequence represented by SEQ ID NO: 1 is derived from xanthinobacterium PAMC25641. The method of claim 11, wherein the lipase has a hydrolytic activity of 60% or more at 5 to 15 ° C. 13. The method of claim 11, wherein the lipase selectively hydrolyzes an acyl group having 4 to 14 carbon atoms. delete

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