KR101891234B1 - Novel protease - Google Patents

Abstract

The present invention relates to a new protease derived from Enterobacteria sp., Which can exhibit a constant activity over a wide range and is expected to solve various problems that may occur when using a low temperature or high temperature protease, And the like.

Description

The novel protease {Novel protease}

The present invention relates to a novel protease derived from Enterobacteria sp. And a method for producing the same.

Protease is an enzyme that is known as a protease that hydrolyzes the peptide bond of a protein to produce a small unit of polypeptide or amino acid.

Protease is an enzyme used in various fields and is used not only for the production of eco-friendly chemical products, foods, cosmetics, but also for a composition for medicinal use. Representative production and purification of protease are mainly performed by culturing various microorganisms and isolating and recovering the protease produced by the microorganisms. For example, the Republic of Korea Patent Application Publication No. 2016-0036194 discloses a Pseudoalteromonas arctica PAMC 21717 and a method for producing the same.

The protease can be classified into a high temperature active protease and a low temperature active protease depending on an activation temperature. In the case of a high temperature active protease, since it has a low activity at a low temperature or a room temperature, energy for raising the temperature of the protease is continuously required to improve the activity of the protease. The low temperature protease is sufficient even at low temperature, but it has the advantage that it does not require additional energy because it has activity and can reduce various side reactions that may occur at high temperature. However, since the activity of the low-temperature protease is rapidly reduced at a high temperature, it is necessary to keep the temperature from rising to a temperature outside the activity range, and the yield of the protease is low because the growth rate of the microorganism producing the low- .

Therefore, it is necessary to study the protease that can maintain its activity without being greatly influenced by temperature and can be obtained at a high production yield.

Korean Patent Laid-Open Publication No. 2016-0036194

The invention Enterobacter bacteria species (Enterobacteria sp .) to maintain a constant activity over a wide temperature range.

The present invention provides a protease derived from Enterobacteria sp. Having an activity at 10 to 70 占 폚.

The present invention provides a protease comprising the amino acid sequence of SEQ ID NO: 1.

The present invention provides a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 1 and a transformation vector comprising the nucleic acid molecule.

The present invention provides a microorganism transformed with a transformation vector comprising a nucleic acid molecule that coats the amino acid sequence of SEQ ID NO: 1, and provides a method for mass-producing the protease of the present invention from the transformed microorganism.

The present invention provides a skin whitening cosmetic composition comprising a protease derived from an Enterobacteria sp.

The protease of the present invention, like other proteases, can maintain a uniform activity over a wide temperature range, but it can solve various disadvantages in the production and use of the low temperature protease and the high temperature protease. The protease of the present invention has a skin whitening effect and can be used in cosmetics for skin care.

Fig. 1 is a cleavage map of a recombinant vector pET28a (+): PAMC25617_prot into which a protease gene is inserted.
Figure 2 shows the phylogenic tree after clustering protease protein sequences of eight strains of homology with the enterobacterial species PAMC 25617.
FIG. 3 shows the positions (A) and (B) of the protease (A) and the purified protease band expressed using SDS-PAGE in (A) (3) and (4) are results obtained by inducing protease expression and then loading, (B), (1) the substance not bound to the column, (2) the pH (3) elution with buffer lowered to pH 5.0, and (4) elution with buffer lowered to pH 4.0.
Fig. 4 shows the result of measuring the activity of the protease according to the temperature.

Hereinafter, the present invention will be described in detail. The terms used in the present specification should be construed as generally understood by a person having ordinary skill in the art unless otherwise defined. It is to be understood that the drawings and embodiments are not intended to limit the scope of the present invention, which is not intended to limit the scope of the present invention. It is not.

The invention Enterobacter bacteria species (Enterobacteria sp .). < / RTI >

The novel protease of the present invention may have activity at a temperature ranging from 0 to 70 ° C, preferably from 10 to 70 ° C. According to one embodiment of the present invention, the novel protease has the highest activity at about 40 ° C., but exhibits activity of at least 60% of the maximum activity over the entire range of 10 to 70 ° C. Therefore, the temperature is not limited from the low temperature to the high temperature And can exhibit constant activity. That is, the protease of the present invention is not limited to a low-temperature or high-temperature protease, and can be widely used throughout the industry.

The novel protease of the present invention is a protease consisting of the amino acid sequence of SEQ ID NO: 1 and is derived from an Enterobacteriaceae.

According to one embodiment of the present invention, the novel protease gene of the present invention can be isolated from the enterobacterial species PAMC 25617 and transformed into various strains and expressed. The enterobacterial species PAMC 25617 is known to be capable of expressing low-temperature active protease as an polar microorganism. It is known that a low-activity protease of the enterobacterial species PAMC 25617 rapidly decreases its activity at high temperatures. However, the protease of the present invention not only exhibits activity at a low temperature, but also exhibits activity above a certain level in a wide temperature range without decreasing its activity even at a high temperature. In addition, it corresponds to a novel protease which is very similar to the protease known so far and does not belong to any one of the low temperature protease and the high temperature protease.

The present invention can provide a nucleic acid molecule encoding a protease comprising the amino acid sequence of SEQ ID NO: 1. The nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 1 preferably comprises SEQ ID NO: 2, but is not limited thereto and is not limited as long as it is a nucleic acid molecule capable of encoding the amino acid sequence of SEQ ID NO:

The present invention provides a transforming vector comprising a nucleic acid molecule encoding a protease comprising the amino acid sequence of SEQ ID NO: 1, and can provide a transformed microorganism transformed with a transformation vector. The transformation vector is preferably a pET-28a (+) vector, but is not limited thereto. The microorganism to be transfected is preferably, but not limited to, bacteria, cyanobacteria or microalgae. For example, a group consisting of Escherichia coli, Spirulina platensis, Chlamydomonas reinhardtii, Haematococcus pluvialis and chlorella vulgaris. Lt; / RTI >

The protease of the present invention can be used for skin cosmetics. According to one embodiment of the present invention, when the cosmetic composition containing the protease derived from the purified enterobacterial species PAMC 25617 is treated on the skin, it has a skin whitening effect and can be used in all of the cosmetic composition for skin care. In addition, the skin whitening effect of the cosmetic composition containing the protease of the present invention is remarkably more than twice as much as the whitening effect of the previously known skin whitening protease.

Hereinafter, embodiments for carrying out the present invention will be described. The present invention is not limited to the above-described embodiments, but may be embodied in many other specific forms without departing from the spirit or essential characteristics thereof.

Enterobacteriaceae  Bell( Enterobacteria sp .) PAMC  25617 derived protease ( PAMC  25617_prot)

Example  1-1. Isolation of Protease Gene

Enterobacteria sp. PAMC 25617 was isolated from the glaciers of Zugspitze (47 ° 25 '16' 'N, 10 ° 59' 07 '') at the German-Austrian border with Germany. Enterobacteria sp. PAMC 25617 was cultured in a Polar and Alpine Microbial Collection (Korea Polar Research Institute, Incheon), and the protease gene was isolated by genomic DNA analysis. 5 g / l of peptone (Becton, Dickinson and company (BD), Franklin Lakes, NJ, USA) for the cultivation of the enterobacterial species PAMC 25617; 3 g / l of malt extract (BD, Franklin Lakes, NJ, USA), 10 g of dextrose (D (+) - Glucose, Junsei, Tokyo, Japan) (BD, Franklin Lakes, NJ, USA) plate was inoculated into 50 ml of the liquid medium using a YM medium of Ph 6.2 (+/- 0.2) / l and cultured at 20 占 폚 for 3 days. Genomic DNA was isolated from isolated strains using a genomic DNA mini kit (Invitrogen, Carlsbad, CA, USA). Genome DNA is analyzed through a method of finding an open reading frame having high homology with a known protease gene and detecting the protease activity by expressing the gene in order to find a new protease from the separated genomic DNA Respectively. For gene sequencing, Miseq sequencing system (Illumina) base analyzer was used. Genomic DNA analysis revealed a 1854 bp long gene (SEQ ID NO: 2) consisting of 617 amino acids (SEQ ID NO: 1) and encoding a protein with a predicted molecular weight of 67101 Da.

Example  1-2. Protease gene Cloning

In order to clone the protease gene found through genomic DNA analysis and expressed, as a forward primer with [5'-ggg att c ca tat g cg cac att gtg gcg aat ta-3 '] and reverse primer [5'-ccg ctc gag tta ctt cac atc acc aca ggt c-3 ']. ' Ca tatg ' of the forward primer is the cleavage site recognized by Nde I restriction enzyme (NEB, Ipswitch, MA, USA) and ' ctc gag ' is the Xho I restriction enzyme (Promega, Fitchburg, Is a cleavage site recognized by the human body. (Kim, H., Choi, J. (2014.9) Effect of Temperature on Growth Rate and Protease Activity of Antarctic Mutants Using Polymerase Chain Reaction (PCR) Microorganisms, Korean Journal of Microbiology and Biotechnology, 42 (3): 293-296.).

As a result of sequencing the isolated protease after PCR, it was confirmed that the similarity with the previously known protease was low (FIG. 2).

Example  1-3. Expression of protease from transformants

For transformation, Escherichia coli preparing coli, E. coli) BL21 (DE) (Invitrogen, Carlsbad, CA, USA), and plasmid pET-28a (+) (Invitrogen , Carlsbad, CA, USA) was used as a vector for gene delivery. The plasmid and the amplified protease gene were digested with Nde I and Xho I restriction enzymes, respectively, and ligase was then treated to introduce the gene into the plasmid. The plasmid into which the gene was introduced was transformed into E. coli BL21 (DE3) host by a known transformation method (Jeong, SW, Seo, HS Kim, MK Choi, 2016.6.1). PprM is necessary for up-regulation of katE1, encoding the major catalase of Deinococcus radiodurans, under unstressed culture conditions. Journal of Microbiology, 54 (6): 426-431.

Plasmid-introduced Escherichia coli BL21 (DE3) was maintained at 37 占 폚 in a medium of Luria Bertani (LB; BD, Franklin Lakes, NJ, USA) and kanamycin (Thermo scientific, Waltham, Mass. USA) was added to the medium and cultured. Protease isopropyl -β-D- thio-galacto-pyrano side of a 1mM concentration of E. coli when the OD ₆₀₀ was about 0.4 to 0.6; the (Isopropyl-β-D-thiogalactopyranoside , IPTG Bio basic, Markham, Ont, Canada) Followed by incubation at 37 ° C for 3 hours and 30 minutes to induce expression.

Example  1-4. Isolation and purification of expressed protease

After expression through IPTG induction, the cell culture was centrifuged at 4,000 x g for 20 minutes to obtain a sample. To isolate the protease protein formed in inclusion body form, , And centrifuged at 4 DEG C at 13000 rpm for 10 minutes to obtain a sample. The obtained sample was resuspended in 8 M concentration of urea, 0.1 M sodium phosphate, 0.01 M Tris-HCl (pH 8.0) and stirred at room temperature for 1 hour. Thereafter, the supernatant was recovered by centrifugation at 4 DEG C and 13000 rpm for 10 minutes.

The supernatant was recovered Ni ^{2 +} -NTA 4 ℃, were separated for 5 minutes and centrifuged at 7000rpm on a column (Elpis biotiech, Dea-jeon, South Korea) the affinity resin. Washed with 8M concentration of urea, 0.1 M sodium phosphate, and 0.01 M Tris-HCl (pH 5.2). Thereafter, the protease was eluted twice using a solution (pH 5.0, pH 4.0) identical to the above composition (Fig. 3 (B)). The concentration of the eluted protein was measured by the method of Bradford ( Anal. Biochem , 72, 248-254, 1976).

After that, the urea was removed by dialysis to allow the recombinant protein to return to its original structure, and the separated recombinant protein was separated by conventional SDS-PAGE (Richard, JS (2004). Purifying proteins for proteomics: a laboratory manual, 1st ed., Cold Spring Harbor Laboratory Press.).

As a result of the separation, the band was observed at a 67 kDa position smaller than 70 kDa, and it was confirmed that the band corresponds to the estimated size from the amino acid sequence containing His ₆ -tag (Fig. 3 (A)).

Measurement of activity of purified protease

A 50 mM concentration containing 10 mM N-succinyl-Ala-Ala-Pro p-nitroanilide (suc-AAPF-pNA) (Sigma-Aldrich, St. Louis, Mo., USA) was used for the measurement of the activity of the enterobacterial species PAMC 25617- Of sodium phosphate (pH 7.2) buffer, and then hydrolyzed at a specific temperature for 10 minutes. Suc-AAPF-pNA was degraded for 10 minutes and the amount of liberated Pna was determined by measuring the absorbance at 410 nm wavelength. One unit of protease activity at this time was determined by the amount of protease enzyme liberating 1 μmole para-nitroanilide equivalent to p-nitroanilide per minute under assay conditions and 8800 as 1% extinction coefficient.

Using the above method, the protease enzyme activity was measured at 10 to 70 ° C in 10 mM Sodium Phosphate Buffer (pH 7.2) and reacted with 10 mM Suc-AAPF-pNA. 40 ° C, and exhibited an activity of 60% or more of the maximum activity over the entire range of 10 to 70 ° C (FIG. 4).

Enterobacteriaceae  Bell PAMC  Measurement of whitening effect of 25617 derived protease

A cosmetic composition containing an enterobacterial species PAMC 25617 derived protease was prepared and its whitening effect was confirmed to be increased. A cosmetic composition was prepared at the composition ratio shown in [Table 1] below.

ingredient Content (w / v%) PAMC 25617 protease 0.5 Lactic acid 1.0 Glycolic acid 1.0 Butylene glycol 2.0 Propylene glycol 6.0 Carboxyvinyl polymer 0.5 Wax 2.0 vaseline 7.0 Polysorbate 60 2.0 Sorbitan sesquioleate 2.5 Squalane 3.0 Liquid paraffin 10.0 Triethanolamine 0.5 Tocopheryl acetate 0.1 Spices 0.05 antiseptic 0.01 Purified water Balance total 100

The whitening effect of the cosmetic composition was evaluated by treating the skin of a woman in his twenties. As a comparative example, a cosmetic composition containing a protease produced by intestinal microbial fermentation of a Korean whiteness spider with a known whitening protease instead of the PAMC 25617 protease shown in Table 1 was prepared and treated. Each arm of the subject was marked by 1 cm in width and length and irradiated with ultraviolet UV-A at a dose of 15 J / cm 2 to cause blackening, and then each of the cosmetic compositions was applied twice a day for one month. Skin color (L value) was measured after 1 month using a skin color measuring device (Minolta CR 300). As a result, the skin color change DL was 1.74 when the cosmetic composition containing the protease derived from the spore moth was treated, but the change DL of the skin color when treated with the cosmetic composition containing the protease derived from the enterobacterial species PAMC 25617 was 3.71 Value, indicating that the whitening effect of the protease derived from the enterobacterial species PAMC 25617 was better.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Novel protease <130> P16080621153 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 617 <212> PRT <213> Enterobacteria sp. PAMC 25617 <400> 1 Met Arg Thr Leu Trp Arg Ile Ile Ala Gly Phe Phe Lys Trp Thr Trp   1 5 10 15 Arg Leu Leu Asn Phe Val Arg Glu Phe Ile Leu Asn Val Phe Leu Ile              20 25 30 Leu Ile Ile Leu Ala Gly Val Gly Ile Trp Tyr Ala Val Gln Asp Lys          35 40 45 Pro Val Asp Thr Thr Lys Gly Ala Leu Leu Val Asp Leu Ser Gly Ser      50 55 60 Val Val Asp Lys Pro Ser Val Asn Asn Lys Val Arg Gln Trp Gly Arg  65 70 75 80 Glu Leu Leu Gly Thr Ser Ser Ser Leu Gln Glu Asn Ser Leu Phe                  85 90 95 Asp Leu Val Asp Thr Leu Arg Ala Ala Lys Asp Asp Lys Asn Val Thr             100 105 110 Gly Ile Val Leu Gln Leu Thr Asp Phe Thr Gly Thr Asp Gln Ala Ser         115 120 125 Met Gln Tyr Ile Gly Lys Ala Leu Arg Glu Phe Arg Asp Ala Gly Lys     130 135 140 Pro Val Tyr Ala Ile Gly Asp Ser Tyr Asn Gln Ser Gln Tyr Tyr Leu 145 150 155 160 Ala Ser Tyr Ala Asn Lys Ile Tyr Met Ser Pro Gln Gly Ala Val Asp                 165 170 175 Leu His Gly Phe Ala Thr Asn Asn Leu Tyr Tyr Lys Ser Leu Leu Glu             180 185 190 Lys Leu Lys Val Thr Thr Asn Ile Phe Arg Val Gly Thr Tyr Lys Ser         195 200 205 Ala Val Glu Pro Leu Ile Arg Asp Asp Met Ser Pro Ala Ala Arg Glu     210 215 220 Ala Asp Ser Arg Trp Ile Gly Gly Leu Trp Thr Asn Tyr Leu Asn Thr 225 230 235 240 Val Ser Ala Asn Arg Gln Ile Thr Pro Glu Gln Leu Phe Pro Gly Ala                 245 250 255 Ala Gly Val Leu Ala Gly Met Gln Ala Thr Gly Asp Met Ala Gln             260 265 270 Tyr Ala Leu Lys Ala Lys Leu Val Asp Ala Leu Ala Ser Arg Thr Glu         275 280 285 Ala Asp Asn Glu Met Val Lys Ala Phe Gly Trp Asn Lys Asp Thr Lys     290 295 300 Asp Phe Asn Tyr Thr Ser Ile Tyr Asp Tyr Ser Pro Lys Pro Lys Pro 305 310 315 320 Asp Asn Asp Pro Gln Ile Ala Val Ile Phe Ala Thr Gly Ala Ile                 325 330 335 Asn Asp Gly Glu Asp Thr Thr Ala             340 345 350 Gln Gln Ile Arg Asp Ala Arg Leu Asp Pro Lys Val Lys Ala Ile Val         355 360 365 Leu Arg Val Val Ser Ser Ser Ser Val Ser Ser Ser Val Val Ser     370 375 380 Arg Ser Glu Leu Ala Ala Aly Lys Ala Ala Gly Lys Pro Val Val Val 385 390 395 400 Ser Met Gly Gly Met Ala Ala Ser Gly Gly Tyr Trp Ile Ser Thr Pro                 405 410 415 Ala Asn Tyr Ile Ile Ala Ser Ser Thr Leu Thr Gly Ser Ile Gly             420 425 430 Ile Phe Gly Val Ile Asn Thr Tyr Gln Asn Thr Leu Asp Tyr Ala Gly         435 440 445 Val His Thr Asp Gly Val Ala Thr Ser Pro Leu Ala Asp Ile Ala Ser     450 455 460 Thr Lys Ala Leu Pro Pro Glu Phe Ser Gln Met Met Gln Leu Asn Ile 465 470 475 480 Glu Asn Gly Tyr Lys Thr Phe Leu Gly Leu Val Ala Asp Ser Arg His                 485 490 495 Lys Thr Pro Glu Glu Ile Asp Gln Ile Ala Gln Gly His Val Trp Ile             500 505 510 Gly Ser Asp Ala Lys Ala Asn Ala Leu Val Asp Glu Leu Gly Asp Phe         515 520 525 Asp Asp Ala Val Lys Lys Ala Ala Asp Leu Ala Lys Leu Pro Lys Trp     530 535 540 Gln Leu Asn Trp Tyr Val Ser Glu Pro Ser Leu Ser Asp Leu Val Phe 545 550 555 560 Ser Gln Val Ser Ala Ser Val His Ala Met Leu Pro Ala Ala Ile Gln                 565 570 575 Ala Tyr Leu Pro Ala Pro Val Ser Lys Met Ala Met Glu Leu Lys Ser             580 585 590 Gln Ala Asp Leu Phe Ser Asn Met Asn Asp Pro Gln Asn Arg Tyr Ala         595 600 605 Leu Cys Leu Thr Cys Gly Asp Val Lys     610 615 <210> 2 <211> 1854 <212> DNA <213> Enterobacteria sp. PAMC 25617 <400> 2 atgcgcacat tgtggcgaat tatcgccggt ttttttaagt ggacctggcg tcttcttaat 60 ttcgtcagag aattcattct caacgttttc cttattctga tcattctggc tggcgtgggc 120 atctggtacg ccgtgcagga caaacccgtc gacaccacca aaggcgcgct gctggttgac 180 ctgagtggtt ccgtggtgga taagccttcg gtgaataata aagtccgtca gtggggccgc 240 gaattactgg gcacgtcgag cagccgtttg caggagaatt ccctgtttga tctggtcgat 300 acccttcgcg ccgcgaaaga tgacaaaaac gtcaccggca ttgttttgca gctgaccgat 360 ttcaccggca ctgaccaggc ctctatgcaa tatattggca aggcgctgcg tgagttccgt 420 gacgcgggta aaccggttta tgccatcggc gacagctata accagtcgca atattatctg 480 gcgagctacg ccaacaaaat ctacatgtca ccacaaggcg cggtggactt gcacggtttt 540 gcgaccaaca atctgtatta caaatccctg cttgagaagc tgaaagtcac gaccaacatt 600 ttccgcgtcg gaacctataa atccgccgtt gaaccgctga tccgtgacga tatgtctcct 660 gcagcacgcg aagcagacag tcgctggatt ggtggattgt ggaccaatta cctcaacacc 720 gtgtcggcaa accgtcagat tacgcctgaa caattgttcc ctggcgcggc gggtgtatta 780 gcgggtatgc aggctacggg cggcgatatg gcgcaatacg cgctgaaagc aaaactggtg 840 gatgcgctgg cttcacgtac cgaagcggat aacgagatgg tgaaagcctt tggctggaat 900 aaagacacca aagactttaa ctacaccagc atctacgatt attcgccgaa accaaaaccg 960 gacaataacg atccgcaaat cgcggtgatc ttcgcgaccg gagcgatcaa tgacggtgaa 1020 gaacaaccgg gtgcagtcgg tggcgacaca accgcgcagc agattcgtga tgcccgtctg 1080 gatccgaaag tgaaagcgat tgtcttacgc gttaacagcc cgggcggcag cgtcagtgcc 1140 tctgaagtga tccgctctga actggctgct gcgaaagccg caggcaaacc agtggtggtt 1200 tccatgggcg gtatggcggc atccggcggt tactggattt caacgccagc gaactacatc 1260 atcgccagcc cgagcaccct caccggctcc atcggtatct ttggcgtgat caacacgtat 1320 cagaacacac tggattacgc aggcgttcac actgacggcg tggcaacatc gccgctggcg 1380 gatattgcct ccaccaaagc gctgcctcct gagttctcgc agatgatgca gctgaacatc 1440 gagaatggtt ataaaacctt cctcggtctg gttgccgact cacgccacaa aacgcctgaa 1500 gaaatcgacc agattgcgca aggccacgtg tggattggtt ctgatgccaa ggccaatgcc 1560 ctggtggatg agctgggtga tttcgatgac gccgtgaaaa aagccgctga tctggcgaag 1620 ctgccgaaat ggcagctgaa ctggtatgtc agcgagccaa gcctgagcga cctggtgttc 1680 tcacaagtga gtgcatccgt tcatgccatg ctgccagcgg cgatccaggc ttatctgcct 1740 gcgccggtga gcaaaatggc gatggagttg aaatcccagg ctgacctgtt cagcaacatg 1800 aatgacccgc aaaaccgtta cgcactttgc ctgacctgtg gtgatgtgaa gtaa 1854

Claims (10)

An enterobacteria sp. PAMC 25617-derived protease having an activity of 80% or more of the maximum activity of the protease at 30 to 60 ° C and consisting of the amino acid sequence of SEQ ID NO: 1.
delete delete delete A nucleic acid molecule encoding the protease of claim 1.
6. The method of claim 5,
Wherein the nucleic acid molecule is a nucleic acid molecule of SEQ ID NO: 2.
A transforming vector comprising the nucleic acid molecule of claim 5 or 6.
A transformed microorganism transformed with the vector of claim 7.
9. The method of claim 8,
The transforming microorganism is a transformed microorganism transformed into Escherichia coli, Spirulina platensis, Chlamydomonas reinhidis, Haematophilus fruticus or Chlorella vulgaris.
A cosmetic composition for skin whitening comprising the protease of claim 1.

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