KR20180027079A

KR20180027079A - Cold-adaptive peptidase from Janthinobacterium sp. and preparation method thereof

Info

Publication number: KR20180027079A
Application number: KR1020160114211A
Authority: KR
Inventors: 최종일; 김현도; 김수미
Original assignee: 전남대학교산학협력단
Priority date: 2016-09-06
Filing date: 2016-09-06
Publication date: 2018-03-14
Also published as: KR101866810B1

Abstract

The present invention relates to a Janthinobacterium sp.-originated low temperature active peptidase and a manufacturing method thereof. The Janthinobacterium sp. PAMC 25641-originated peptidase according to the present invention solves a problem of a side reaction caused by a high temperature pretreatment process and a reaction at high temperatures, thereby expecting to be widely used in food, drug and biotechnology industries.

Description

[0002] Cold-adapted peptidase from Janthinobacterium sp. and preparation method thereof}

본 발명은 잔티노박테리움 sp.(Janthinobacterium sp.) 유래 저온 활성 펩티다아제 및 그 제조방법에 관한 것이다.The present invention relates to a low-temperature active peptidase derived from Janthinobacterium sp. And a method for producing the same.

펩티다아제(peptidase)는 단백질(protein)과 펩티드(peptide)에서 펩티드 결합(peptide bond)을 가수분해하는 효소이다. 펩티다아제는 전 세계 효소 산업의 약 60%를 차지할 정도로 산업 분야에서 중요한 요소이며, 세제, 가죽, 식품산업, 약품 및 생물학적 환경 정화 등 다양한 분야에서 활용되고 있다. 지난 몇 년 간, 고온에서 활성이 있는 펩티다아제에 대한 많은 연구가 이루어졌지만, 고온 활성이 있는 펩티다아제는 저온이나 일반적인 효소가 활성을 나타내는 온도에서 낮은 활성을 보인다는 단점이 있다. 그러나, 저온에서 활성을 나타내는 펩티다아제는 부수적인 열처리의 필요성이 없어 에너지 및 시간 절약이 가능하고 특히 낮은 온도에서 효과적으로 독성 물질의 영향 감소가 가능하여 최근에 주목을 받고 있다.Peptidases are enzymes that hydrolyze peptide bonds in proteins and peptides. Peptidase is an important element in the industrial sector, accounting for about 60% of the world's enzymes industry, and is used in a variety of fields including detergents, leather, food industry, pharmaceuticals and biological environment purification. Over the past several years, many studies have been made on peptidases that are active at high temperatures, but peptidases with high temperature activity have a disadvantage of low activity at low temperatures or at temperatures at which general enzymes are active. However, peptidases exhibiting activity at low temperatures have been recently attracting attention because they do not require an additional heat treatment and thus can save energy and time, and can effectively reduce the effects of toxic substances particularly at low temperatures.

근래 아시네토박터 sp.(Acinetobacter sp.), 알테로모나스 sp.(Alteromonas sp.) 및 슈도모나스 sp.(Pseudomonas sp.)에서 유래한 펩티다아제를 정제하여 특성화한 연구 결과가 보고된 바 있다.Recently Acinetobacter sp. (Acinetobacter sp.), Alteromonas sp. (Alteromonas sp.), And Pseudomonas sp. (Pseudomonas sp.) Have been purified to characterize the resulting peptidase results are reported in.

효소를 높은 수율로 얻기 위해서는 원핵생물(prokaryotes)을 숙주로 이용하여 유전자 형질전환 및 발현을 통하는 방법이 관심을 받고 있다. 그러나, 저온 활성 펩티다아제에 대한 유전자 형질전환, 발현 및 정제에 대한 연구는 미흡한 실정이다. 따라서 상기 문제를 해결하기 위해 고수율로 저온 활성 펩티다아제를 생산할 수 있는 기술의 개발이 절실한 상황이다.In order to obtain the enzyme at a high yield, a method of using the prokaryotes as a host to transfect and express the gene is of interest. However, studies on gene transfection, expression and purification against low temperature active peptidase are insufficient. Therefore, it is inevitable to develop a technology capable of producing a low-temperature active peptidase at a high yield in order to solve the above problem.

대한민국 공개특허공보 2009-0005599호.Korean Patent Publication No. 2009-0005599. 대한민국 공개특허공보 2014-0127957호.Korean Patent Publication No. 2014-0127957.

Choi J et al. Theories and Application of Chemical Engineering., Vol. 22, No. 1, 2016. Choi J et al. Theories and Application of Chemical Engineering., Vol. 22, No. 1, 2016.

상기와 같은 문제점의 해결을 위해 본 발명에서는 저온 활성 균주인 잔티노박테리움 sp.(Janthinobacterium sp.)로부터 저온 활성 펩티다아제를 발현 정제한 후, 유전자 서열을 밝히고, 이 유전자를 분리하여 형질전환을 통한 발현을 통해 저온에서 효소 활성을 나타냄을 확인하고자 하였다.In order to solve the above problems, the present invention provides a method of expressing and purifying a low-temperature active peptidase from a low-temperature active strain, Janthinobacterium sp., And identifying the gene sequence, isolating the gene, And the expression of the enzyme at low temperature.

본 발명은 서열번호 1의 아미노산 서열을 갖는 잔티노박테리움 sp.(Janthinobacterium sp.) PAMC 25641 유래 펩티다아제(peptidase)에 관한 것이다.The present invention relates to a peptidase derived from Janthinobacterium sp. PAMC 25641 having the amino acid sequence of SEQ ID NO: 1.

또한 본 발명은 상기 펩티다아제를 코딩하는 핵산 분자에 관한 것이다.The present invention also relates to a nucleic acid molecule encoding said peptidase.

또한 본 발명은 상기 핵산 분자를 포함하는 벡터(vector)에 관한 것이다.The present invention also relates to a vector comprising the nucleic acid molecule.

또한 본 발명은 상기 벡터로 형질전환된, 인간을 제외한 형질전환체에 관한 것이다.The present invention also relates to a transformant transformed with said vector, excluding human.

또한 본 발명은 상기 핵산 분자를 포함하는 펩티다아제 유전자가 삽입된, 도 5의 개열지도를 갖는 재조합 벡터 pET-28a(+):PAMC25641_pep에 관한 것이다.The present invention also relates to a recombinant vector pET-28a (+): PAMC25641_pep having the cleavage map of FIG. 5 inserted with a peptidase gene comprising the nucleic acid molecule.

본 발명을 통해 얻은 잔티노박테리움 sp. PAMC 25641 유래 펩티다아제는 고온 전처리 과정과 고온에서의 반응으로 인해 야기되는 부반응 문제를 해결함으로써, 식품, 약품 및 생물 산업 등에 널리 활용될 수 있을 것으로 기대된다.The Zanthinobacterium sp. The peptidase derived from PAMC 25641 is expected to be widely used in food, medicine, and biotechnology industries by solving the side reaction problem caused by the high-temperature pretreatment process and the reaction at a high temperature.

도 1은 배양한 잔티노박테리움 sp. PAMC 25641의 상등액을 0 내지 100%의 각각 다른 포화도의 황산암모늄(ammonium sulfate)을 첨가하여 침전시켰을 때 나타난 펩티다아제 활성을 측정한 결과이다.
도 2는 DEAE-sepharose 컬럼으로 용출된 시료에서 펩티다아제 활성을 측정한 결과이다.
도 3은 펩티다아제 온도(A) 및 pH(B)에 따른 활성을 측정한 결과이다.
도 4는 SDS-PAGE를 이용하여, 발현된 펩티다아제 밴드 위치를 표시한 것으로 각 레인은 (1) pET28a, (2) 0.1 mM IPTG 첨가 전, (3) 0.1 mM IPTG 첨가 후 생성된 세포 내의 단백질 및 (4) 정제된 단백질 로딩 결과이다.
도 5는 펩티다아제 유전자가 삽입된 재조합 벡터 pET-28a(+):PAMC25641_pep의 개열지도를 나타낸 것이다.Fig. 1 shows the results of cultured Zanthinobacterium sp. PAMC 25641 was precipitated by the addition of 0 to 100% of ammonium sulfate at different degrees of saturation, respectively.
FIG. 2 shows the result of measuring the peptidase activity in a sample eluted with a DEAE-sepharose column.
FIG. 3 shows the results of measuring the activity according to the peptidase temperature (A) and the pH (B).
FIG. 4 shows the positions of the expressed peptidase bands using SDS-PAGE. Each lane shows (1) pET28a, (2) before addition of 0.1 mM IPTG, (3) (4) purified protein loading results.
5 shows a cleavage map of a recombinant vector pET-28a (+): PAMC25641_pep into which a peptidase gene is inserted.

본 발명에서 상기 핵산 분자는 특별히 제한되지는 않지만 서열번호 2의 염기 서열을 갖을 수 있다.In the present invention, the nucleic acid molecule is not particularly limited, but may have the nucleotide sequence of SEQ ID NO: 2.

본 발명에서 상기 형질전환체는 특별히 제한되지는 않으나 박테리아, 시아노박테리아 또는 미세조류일 수 있다.In the present invention, the transformant is not particularly limited, but may be bacteria, cyanobacteria or microalgae.

이하, 본 발명의 내용을 실시예를 통하여 보다 구체적으로 설명한다. 이들 실시예는 본 발명의 내용을 이해하기 위해 제시되는 것일 뿐 본 발명의 권리범위가 이들 실시예로 한정되는 것은 아니고, 당업계에서 통상적으로 주지된 변형, 치환 및 삽입 등을 수행할 수 있으며, 이에 대한 것도 본 발명의 범위에 포함된다.Hereinafter, the content of the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited to these embodiments and that variations, substitutions, and insertions conventionally known in the art can be carried out, And this is included in the scope of the present invention.

[펩티다아제 활성 측정 방법][Method for measuring peptidase activity]

잔티노박테리움 sp. PAMC 25641로부터 생산된 펩티다아제의 활성도는 10 mM N-Succinyl-Ala-Ala-Pro p-nitroanilide (Suc-AAPF-pNA; Sigma-Aldrich, St. Louis, MO)가 포함된 50 mM 인산 나트륨(pH 7.2) 완충용액에서, 25 ℃에서 10 분간 상기 Suc-AAPF-pNA가 분해될 때 유리되는 pNA의 양을 흡광도 410 nm에서 측정하여 결정하였다.Zanthinobacterium sp. The activity of the peptidase produced from PAMC 25641 was measured using 50 mM sodium phosphate (pH 7.2) containing 10 mM N-Succinyl-Ala-Ala-Pro-nitroanilide (Suc-AAPF-pNA; Sigma-Aldrich, St. Louis, Mo.) ) Buffer solution, the amount of pNA liberated when the Suc-AAPF-pNA was degraded at 25 캜 for 10 minutes was determined by measuring the absorbance at 410 nm.

이 때, 펩티다아제 활성의 1 단위(unit)는 상기 조건에서 분당 파라-니트로 아닐라이드(p-nitroanilide)와 등가의 1 μmole 파라-니트로 아닐라이드를 유리시키는 펩티다아제 효소의 양으로 결정하였고, 1 % 흡광계수로 8800을 이용하였다.At this time, one unit of peptidase activity was determined by the amount of peptidase enzyme liberating 1 탆ole para-nitroanilide equivalent to p-nitroanilide per minute under the above conditions, and 1% absorbance 8800 was used as a coefficient.

[실시예 1] 잔티노박테리움 sp. PAMC 25641의 배양[Example 1] Preparation of Zanthinobacterium sp. Culture of PAMC 25641

잔티노박테리움 sp.(Janthinobacterium sp.) PAMC 25641 균주는 사우스셰틀랜드 제조(South Shetland Islands)의 킹조지 섬(King George Island, 62˚13.18' S, 58˚47.20' W) 근해로부터 분리되었다. Janthinobacterium sp. PAMC 25641 strain was isolated from the coast of King George Island (62 ° 13.18 'S, 58 ° 47.20' W), South Shetland Islands.

10 g/L 덱스트로스(Junsei, Tokyo, Japan), 5 g/L 펩톤(Becton, Dickinso and Company, New Jersey, USA) 및 0.1 g/L 트리톤 X-100 (pH 4.8; Duksan Pure Chemicals, 대한민국)로 구성된 배지를 이용하여 한천(Becton, Dickinso and Company, New Jersey, USA) 플레이트에 형성된 단일 콜로니를 50 mL 액체 배지에 접종한 후 15 ℃에서 96 시간동안 배양하였다.L of Triton X-100 (pH 4.8; Duksan Pure Chemicals, Korea), 10 g / L Dextrose (Junsei, Tokyo, Japan), 5 g / L peptone (Becton, Dickinson and Company, New Jersey, USA) (Becton, Dickinson and Company, New Jersey, USA) plates were inoculated into 50 mL liquid medium and cultured at 15 DEG C for 96 hours.

[실시예 2] 잔티노박테리움 sp. PAMC 25641에서 얻은 펩티다아제 정제[Example 2] Preparation of Zanthinobacterium sp. Peptidase purified from PAMC 25641

상기 배양을 통해 생성된 펩티다아제 정제를 위해 배양 상등액 1 L에 0 내지 100%의 각각 다른 포화도의 황산암모늄(ammonium sulfate)을 첨가하여 침전시킨 후 4 ℃, 21,000 x g에서 30분 동안 원심분리하였다.For purification of the peptidase produced through the above cultivation, 1 L of culture supernatant was added with 0 to 100% of different saturation ammonium sulfate, and then centrifuged at 21,000 x g for 30 minutes at 4 ° C.

20 mM Tris-HCl(pH 8.5; Sigma-Aldrich,St. Louis, MO) 완충용액에 침전된 펠렛(pellet)을 용해시키고, 동일 완충 용액에서 투석한 후 상기 펩티다아제 활성 측정 방법을 통해 활성을 측정하였다.The pellet precipitated in a buffer solution of 20 mM Tris-HCl (pH 8.5; Sigma-Aldrich, St. Louis, MO) was dissolved and dialyzed in the same buffer solution, and the activity was measured by the peptidase activity measurement method .

그 결과, 도 1에서 확인할 수 있는 바와 같이 30 ~ 80% 범위의 포화도에서 펩티다아제의 활성이 확인되었고, 펩티다아제 침전의 최적 포화도는 60 ~ 70%인 것으로 나타났다.As a result, as shown in FIG. 1, peptidase activity was confirmed at a degree of saturation ranging from 30 to 80%, and the optimum degree of saturation of the peptidase precipitation was 60 to 70%.

상기 침전을 통해 얻은 펩티다아제를 20 mM Tris-HCl(pH 8.5; Sigma-Aldrich,St. Louis, MO) 완충용액dp 의해 평형화된 DEAE-sepharose 컬럼(GE Healthcare, Little Chalfont, UK)에 처리하였고, 펩티다아제는 0 ~ 0.5 M 염화나트륨 농도 구배에 따라 30 mL/시간의 속도로 10 mL씩 용출되었다. 용출도니 단백질의 농도는 브래드포드(Bradford M., Anal BioChem, 72, 248-254, 1976)의 방법으로 측정하였고, 용출된 각 샘플을 사용하여 펩티다아제 활성을 측정하였다.The peptidase obtained from the precipitation was treated on a DEAE-sepharose column (GE Healthcare, Little Chalfont, UK) equilibrated with dp of 20 mM Tris-HCl (pH 8.5; Sigma-Aldrich, St. Louis, MO) buffer, Was eluted with 10 mL each at a rate of 30 mL / hour according to a gradient of 0 to 0.5 M sodium chloride concentration. The concentration of the eluted protein was determined by the method of Bradford ( Anal. Bio . Chem. , 72, 248-254, 1976) and the eluted samples were used to determine the peptidase activity.

그 결과, 도 2에서 확인할 수 있는 바와 같이 70 ~ 120 mM 염화나트륨에서 용출된 샘플에서 펩티다아제 활성이 측정되었고, 가장 높은 효소 활성을 나타낸 90 mM 염화나트륨에서 용출된 샘플을 이용하여 생화학적 특성을 분석하였다.As a result, peptidase activity was measured in a sample eluted from 70 to 120 mM sodium chloride as shown in FIG. 2, and biochemical characteristics were analyzed using a sample eluted from 90 mM sodium chloride showing the highest enzyme activity.

각 펩티다아제 정제 단계에 따른 활성 및 수율을 표 1에 나타내었다.The activity and the yield according to each peptidase purification step are shown in Table 1.

[표 1] 펩티다아제 활성 및 수율[Table 1] Peptidase Activity and Yield

[실시예 3] 정제된 펩티다아제의 생화학적 특성[Example 3] Biochemical characteristics of purified peptidase

온도 변화에 따른 활성Activity by temperature change

상기 펩티다아제 활성 측정 방법을 통해 5 내지 60 ℃ 범위에서 펩티다아제 활성을 측정한 결과, 상기 넓은 온도 범위에서도 활성을 나타내었으며, 도 3에서 확인할 수 있는 바와 같이 40 ℃에서 가장 높은 활성을 나타내는 것을 확인하였다.As a result of measuring the peptidase activity in the range of 5 to 60 ° C by the above peptidase activity measurement method, the peptide activity was exhibited even in the wide temperature range, and it was confirmed that the peak activity was shown at 40 ° C as shown in FIG.

pH 변화에 따른 활성Activity by pH change

상기 펩티다아제 활성 측정 방법을 통해 하기 표 2와 같이 4 내지 10까지 pH를 변화시키며 활성을 측정하였다.The peptidase activity was measured by varying the pH from 4 to 10 as shown in Table 2 below.

[표 2] pH 변화에 따른 활성[Table 2] Activity due to pH change

그 결과 도 3과 같이, pH 7.5에서 가장 높은 활성이 나타났고, pH 9.5에서 활성이 급격하게 저하되는 것을 확인하였다.As a result, as shown in Fig. 3, the highest activity was observed at pH 7.5, and the activity was abruptly decreased at pH 9.5.

이온 또는 저해제 첨가에 따른 활성Activity due to ion or inhibitor addition

상기 펩티다아제 활성 측정 방법을 통해 하기 표 3과 같이 금속 이온 또는 저해제를 처리하면서 효소의 활성을 측정하였다. The activity of the enzyme was measured by treating the metal ion or the inhibitor with the peptidase activity measuring method as shown in Table 3 below.

[표 3] 금속 이온 또는 저해제 첨가에 따른 펩티다아제 활성[Table 3] Peptidase activity by addition of metal ion or inhibitor

그 결과 상기 표에서와 같이 10 mM Mn²⁺, Fe³⁺, Cu²⁺, Zn²⁺ 금속 이온을 처리하였을 때, 효소 활성이 대조군의 10% 이하로 감소하였고, 1 mM PMSF을 처리한 경우 효소 활성이 대조군의 45% 이하로 감소하였다.As a result, when the 10 mM Mn ²⁺ , Fe ³⁺ , Cu ²⁺ , and Zn ²⁺ metal ions were treated, the enzyme activity was reduced to 10% or less of that of the control group, Enzyme activity decreased to less than 45% of the control.

반면, 10 mM K⁺, Na⁺를 처리한 경우 효소 활성이 대조군에 비해 각각 18%, 15% 향상되는 것을 확인하였다.On the other hand, enzyme activity was improved by 18% and 15% when 10 mM K ⁺ and Na ⁺ were treated, respectively.

[실시예 4] 잔티노박테리움 sp. PAMC 25641 유래 펩티다아제(PAMC25641_pep) 유전자 분리, 형질전환 및 발현[Example 4] Preparation of Zanthinobacterium sp. PAMC 25641-derived peptidase (PAMC25641_pep) gene isolation, transformation and expression

펩티다아제 유전자 분리Peptidase gene isolation

실시예 1과 동일한 방법으로 잔티노박테리움 sp PAMC 25641 균주를 배양한 후, 게노믹 DNA 미니 키트(Genomic DNA Mini Kit; Invitrogen, CA)를 이용하여 게놈 DNA(genomic DNA)를 분리하였다.Genomic DNA (genomic DNA) was isolated using Genomic DNA Mini Kit (Invitrogen, Calif.) After culturing Zanthinobacterium sp. PAMC 25641 strain in the same manner as in Example 1.

상기 게놈 DNA로부터 생물정보학 기법을 사용한 유전자 검색을 통해(NCBI ORF Finder tool (http://www.ncbi.him.nih.gov/gorf/gorf/gorf.html) 및 InterProtool (https://www.ebi.ac.uk/interpro/) 1,054 개의 아미노산 서열을 갖으며, 예측 분자량이 106,630 Da인 단백질을 코딩하는 3,165 개의 염기서열을 갖는 유전자를 발견하였다.From the genomic DNA, gene search using bioinformatics techniques ( NCBI ORF Finder tool (http://www.ncbi.him.nih.gov/gorf/gorf/gorf.html) and InterProtool (https: // www. ebi.ac.uk/interpro/) found a gene with 3,165 nucleotide sequences coding for a protein with 1,054 amino acid sequences and a predicted molecular weight of 106,630 Da.

펩티다아제 유전자 클로닝 및 형질전환Peptidase gene cloning and transformation

상기 유전자를 발현시키기 위해, 하기 표 4의 프라이머를 사용하였다.In order to express the gene, the primers shown in Table 4 were used.

정방향 프라이머에서 굵은 밑줄 글씨는 NdeI 제한효소 (NEB, Ipswitch, MA)가 인식하는 절단 부위, 역방향 프라이머에서 굵은 글씨는 HindIII 제한효소 (NEB, Ipswitch, MA)가 인식하는 절단 부위를 나타낸다.In the forward primer, the bold underline indicates the cleavage site recognized by the Nde I restriction enzyme (NEB, Ipswitch, MA), and the bold in the reverse primer indicates the cleavage site recognized by the Hind III restriction enzyme (NEB, Ipswitch, MA).

[표 4] 프라이머 및 제한효소 절단부위[Table 4] Primer and restriction enzyme cleavage site

상기 프라이머 쌍을 이용하여 통상적인 중합효소연쇄반응(polymerase chain reaction, PCR) 방법으로 유전자 증폭을 수행하였다.Gene amplification was performed using a conventional polymerase chain reaction (PCR) method using the above primer pairs.

형질전환을 위한 숙주로 대장균(Escherichia coli) BL21(DE3) (Invitrogen, CA)을, 유전자 전달을 위한 벡터로 플라스미드 pET-28a(+) (Invitrogen, CA)를 사용하였다. Escherichia coli BL21 (DE3) (Invitrogen, CA) was used as a host for transformation and plasmid pET-28a (+) (Invitrogen, CA) was used as a vector for gene transfer.

상기 플라스미드 pET-28a(+)와 증폭된 유전자를 각각 NdeI 제한효소와 HindIII제한효소로 전달한 후, 리가아제(ligase; NEB, Ipswitch, MA)로 플라스미드에 유전자를 접합하여 제조하였다. 이후 상기 플라스미드를 통상적인 전기충격(electronic shock) 방법을 사용하여 상기 대장균에 형질전환하였다.The plasmid pET-28a (+) and the amplified gene were transferred to Nde I restriction enzyme and Hind III restriction enzyme, respectively, and ligated to the plasmid with ligase (NEB, Ipswitch, MA). The plasmid was then transformed into E. coli using a conventional electronic shock method.

펩티다아제 유전자 발현Peptidase gene expression

상기 형질전환된 대장균을 37 ℃가 유지되는 루리아-버타니(Luria-Bertani, LB; Becton, Dickinso and Company, NJ) 배지에 카나마이신(Kanamycin, Km; Thermo Scientific, MA) 30 μg/mL로 첨가한 후 배양하였다.The transformed Escherichia coli was added to 30 μg / mL kanamycin (Km; Thermo Scientific, MA) in Luria-Bertani (LB; Becton, Dickinson and Company, And then cultured.

대장균의 600 nm 광밀도(optical density, OD)가 0.4 내지 0.6 일 때 0.1 mM 농도의 이소프로필-β-D-티오갈락토피라노사이드 (Isopropyl-β-D-thiogalactopyranoside, IPTG; Biobasic, Ontario, Canada)를 첨가한 후, 20 ℃에서 12 시간동안 배양하여 펩티다아제 단백질을 발현하시켰다.D-thiogalactopyranoside (IPTG; Biobasic, Ontario, USA) at a concentration of 0.1 mM when the optical density (OD) at 600 nm of E. coli was 0.4 to 0.6. Canada), and then cultured at 20 ° C for 12 hours to express the peptidase protein.

발현된 펩티다아제 단백질의 분리정제Separation purification of the expressed peptidase protein

상기 IPTG 유도 배양을 통해 얻은 세포 배양액을 4 ℃, 4,000 x g에서 20 분간 원심분리하여 샘플을 수득한 후, B-PER bacterial protein extraction reagent(Thermo Scientific, MA)와 라이소자임(lysozyme; Thermo Scientific, MA)을 이용하여 재현탁한 후 20 분 동안 상온에 방치하고 초음파 분해(sonication)를 수행하여 세포를 용해시켰다. 이후, 4 ℃, 13,000 x g에서 10 분간 원심분리하여 상등액을 수득하였다.The cell culture obtained from the IPTG induction culture was centrifuged at 4,000 xg for 20 minutes to obtain a sample. The B-PER bacterial protein extraction reagent (Thermo Scientific, MA) and lysozyme (Thermo Scientific, MA) And the cells were allowed to stand at room temperature for 20 minutes and sonicated to dissolve the cells. Thereafter, the supernatant was obtained by centrifugation at 13,000 x g for 10 minutes at 4 ° C.

수득한 상등액을 1 x PBS 완충용액(phosphate-buffered saline; Thermo Scientific, MA)에 의해 평형화된 Ni²⁺-NTA 친화성 레진 컬럼(Ni²⁺- nitrilotriacetic acid affinity column; Thermo Scientific, MA)에 처리하고, 75 mM 이미다졸을 이용하여 펩티다아제 단백질을 용출시켰다.The obtained supernatant was the 1 x PBS buffer (phosphate-buffered saline; Thermo Scientific , MA) with Ni ²⁺ -NTA column equilibrated affinity resin by; treatment ^{(Ni 2+ nitrilotriacetic acid affinity column Thermo} Scientific, MA) And 75 mM imidazole was used to elute the peptidase protein.

용출된 단백질의 농도는 브래드포드(Bradford M., Anal BioChem, 72, 248-254, 1976)의 방법으로 측정하였고, 용출된 샘플을 사용하여 정제된 펩티다아제 활성을 측정하였다.The concentration of the eluted protein was measured by the method of Bradford (Bradford M., Anal Bio Chem , 72, 248-254, 1976), and the eluted sample was used to measure the purified peptidase activity.

그 결과 도 4에서 확인할 수 있는 바와 같이, 100 kDa 위치에서 밴드가 관측되어 His₆-tag(histidine six-tag)를 포함하는 아미노산 서열로부터 추정된 크기에 해당하는 것을 확인할 수 있었다.As a result, as shown in FIG. 4, the band was observed at the 100 kDa position, and it was confirmed that the band corresponds to the estimated size from the amino acid sequence containing His ₆ -tag (histidine six-tag).

정제된 펩티다아제 활성도을 상기 방법을 통해 측정한 결과 23 U/L을 나타내는 것을 밝혀냈다.The purified peptidase activity was found to be 23 U / L as measured by the above method.

상기로부터, 본 발명의 잔티노박테리움 sp. PAMC 25641 으로부터 분리하여 대장균으로 형질전환을 통해 발현한 단백질이 펩티다아제로서 고온처리에 따른 부반응을 해결하고 여전히 좋은 활성을 나타내고 있음을 발견하여 본 발명을 완성하였다.From the above, it can be seen that the microorganism belonging to the genus Zantinobacterium sp. The present inventors have found that a protein expressed by transformation from PAMC 25641 and transformed into Escherichia coli is a peptidase that solves side reactions caused by high temperature treatment and still exhibits good activity.

<110> University Industry Liaison Office Of Chonnam National University <120> Cold-adaptive peptidase from Janthinobacterium sp. and preparation method thereof <130> P16070971042 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 3165 <212> DNA <213> Artificial Sequence <220> <223> PAMC25641_pep DNA <400> 1 atgaaactac gtcccgtttc actcggtatc ctcttgctgg ccgccagcct ggcgcaagcg 60 gcgcaggcgc aggaacgccg ctcctacatc gtgcagctgg tcgacaagcc ggccgccacc 120 tacacgggcc aggtcgacgg cctggccgcc accaagccgg ccccgggggc acgcatcaac 180 gtgggcgccg ccgacgtgca ggcctatttg aattacctcg acaccagaca ggccgccgtg 240 gccggtaccg tcagcgcggc ggaaatcacg caccggtaca gcgtcgtctt caacggcttt 300 tccgccctgc tgacggacga tgaagtgcgg gcgctgaaga aaaacagcgg cgtggccagc 360 atcagcgccg actccatcct gcaactcgac accagctaca cgcccagctt cctgggcctg 420 gacaaaccgg gtggcatctg ggagcagctg ggcggcaagg cgcatgcggg tgaagacatc 480 atcatcggca tcgtcgacag cggcatctgg cccgagaaca cggcctttgc cgaccgcctc 540 gacgagagcg gcgtgcccag ccacagcggc aacaacgtcg tgtacggcgc gccaccggcc 600 aactggcagg gcatgtgcca gacgggcgaa ggttttggcg ccgagaattg caacaataag 660 ctgatcggtg cgcgctacta ccgcgccgcc acgtcggcgc tgcactggac ggaatttttg 720 tcgccgcgcg attccgtcgc cggcctgacg gggcacggcg gccatggcac gcacacggcg 780 tccacggccg gcggcaacaa tggcgccctg gccagctcga acggtgtgtc gctgggcaag 840 gcgtcgggca tggcgccgcg cgcccgcatc gccgcctata aagtctgctg gaccgccgcc 900 tcgacggggc gcaacggctg cgccacggcc gacagcgtgg ccgccatcga ccaggcggtc 960 aaggatggcg tcaacgtcat caacttttcc atcggcccga acgcgggcgg cggcgccttc 1020 gacgaaccga cggaagtcgc tttcctcggc gcggcggcgg ccggcgtgtt cgtcgccacg 1080 tcgggcggca attccggccc ggccacgccc gcgcccgacg tgccggcgcc cgtgtcgcac 1140 atcagcccgt ggctggcgac ggtggccaat tccacccaca accgcctgta cgcgggcaat 1200 gtgatactca gcaacggcgc caagctggaa ggcgcatcga gcaatgcgaa gacgccggcg 1260 ctgccgctga tccgctcgcg cgacgcgggc ctggccggcg tgtcgccaac ggatgtcaac 1320 ctgctgcgct gcttcggcgc ggccgatacc acctcggcct acctggaccc ggcgaaagtg 1380 gcgggcaaga tactcgtgtg cgaccgcggt ggcaacgtgc tggtcaacaa gagcgcgaat 1440 gccaaaacgg cgggcgcggc gggcgtgatc atcgccaacg tggccggtgg cgccaacacc 1500 atcatcaacc aggcgcacgt gctgtcgacc gtgcacctgg cgcaggcgca gggcgatgcg 1560 ttgaaggctt tcatggcggt caaccccgat ggcacggcgg cattgggcga aatccatacc 1620 atccccgaca cgacggtgca ggcgccgatc gtcagcgacc gttcctcgcg cgggccgaac 1680 gtggccaacg cgaacatcct gaaacccgac ctgtcggccc ccggcaccaa cgtgctggcc 1740 ggcgtgacgg ccgacctgac gccggcgcag cgcgatgccg tggcggccgg cggcgtggcg 1800 cccgtggccg agtgggattt ctattccggc acctcgatgg ccagcccgca cgtggcgggc 1860 gtggcggcat tgctcaagca gcagcacccg acctggtcgc ccgcggccat caagtcggcg 1920 ctgatgacga cggccttcag cacgtattcc gacggcttga acggttccgt gtcgtgggat 1980 gccacggcga agaactcggg ccagttgccg tggggccagg gcgccggcca catcgcgccg 2040 aacagcgcgg ccgatcccgg cctcgtgtac gacgtgtcgg aactcgacta tgcgcgcttc 2100 ctgtgcggcc tgaacctgaa ggtctacagt cccgccacct gccagtctgt cggcaccatt 2160 cccgcctaca acctgaacct ggcgtcgctg acggcggcca acgtgctggg cacgcagaca 2220 ttgacgcgca cggtgaccaa cgtgggcgcc ggcagcgccg tctacaacgt ctcggccagc 2280 ctgcccggct acacggtggc ggtgacgcca acgagtctga gcctggcgcc gggcgccagg 2340 gcgcagtacc aggtcaagct gacgcgcacc acggcgccgg ccaatacctg gacctacggc 2400 gccctgagct ggagcgatgg cacgcacacg gtgcgcagcc cgctgacggc gcgcggcacc 2460 tcgctggccg ccattccact ggtcagcagc gaagcggcca cgggcagcaa ggtgctgacc 2520 ctgggcacgg gcttcacggg cgcgctggtg ggcgtcaagt cggggctggt cgaagccgta 2580 cgtgaagcgc gcacgatcgg ccaggcgacg acgggtgctg ccgcatcggc cgcctgcaag 2640 gccggtggcg ccacgggcgt gaacgtgcac aacgtggtga tcccggccgg caccctggcg 2700 gcgcgctttg ccacctacga cggggaaacg acgggcggcg cgaataccga catggacatg 2760 gaggtctaca acgccgccaa tgtgctggtc ggcagcagcg gcaatgaagg ttccaatgag 2820 caagtggaac tgcgcctgcc cgccgccggc acgtacaagg tgtgcgtgat cggctacgcg 2880 ccgcaaaacg gccaggccga ctacactttg tcgtcgtggg tgctggcgcc gggcctgtcg 2940 aatggcggct tcaaggcgtt gatgccgggc accgcctaca cgggcggcac ggcgacggtc 3000 tcgctgagct ggtccaacct ggtcgaaggc aagcgctacc tgggcgccgt cggctaccaa 3060 gtgggcggcg tggtgcaggg cgtgacggtg gtcgaggtca ataccaatga ccccgtgccg 3120 ctgttccaga acgcgcgcgg cgccaggccg gtgctggctg aataa 3165 <210> 2 <211> 1054 <212> PRT <213> Artificial Sequence <220> <223> PAMC25641_pep Protein <400> 2 Met Lys Leu Arg Pro Val Ser Leu Gly Ile Leu Leu Leu Ala Ala Ser 1 5 10 15 Leu Ala Gln Ala Ala Gln Ala Gln Glu Arg Arg Ser Tyr Ile Val Gln 20 25 30 Leu Val Asp Lys Pro Ala Ala Thr Tyr Thr Gly Gln Val Asp Gly Leu 35 40 45 Ala Ala Thr Lys Pro Ala Pro Gly Ala Arg Ile Asn Val Gly Ala Ala 50 55 60 Asp Val Gln Ala Tyr Leu Asn Tyr Leu Asp Thr Arg Gln Ala Ala Val 65 70 75 80 Ala Gly Thr Val Ser Ala Ala Glu Ile Thr His Arg Tyr Ser Val Val 85 90 95 Phe Asn Gly Phe Ser Ala Leu Leu Thr Asp Asp Glu Val Arg Ala Leu 100 105 110 Lys Lys Asn Ser Gly Val Ala Ser Ile Ser Ala Asp Ser Ile Leu Gln 115 120 125 Leu Asp Thr Ser Tyr Thr Pro Ser Phe Leu Gly Leu Asp Lys Pro Gly 130 135 140 Gly Ile Trp Glu Gln Leu Gly Gly Lys Ala His Ala Gly Glu Asp Ile 145 150 155 160 Ile Ile Gly Ile Val Asp Ser Gly Ile Trp Pro Glu Asn Thr Ala Phe 165 170 175 Ala Asp Arg Leu Asp Glu Ser Gly Val Pro Ser His Ser Gly Asn Asn 180 185 190 Val Val Tyr Gly Ala Pro Pro Ala Asn Trp Gln Gly Met Cys Gln Thr 195 200 205 Gly Glu Gly Phe Gly Ala Glu Asn Cys Asn Asn Lys Leu Ile Gly Ala 210 215 220 Arg Tyr Tyr Arg Ala Ala Thr Ser Ala Leu His Trp Thr Glu Phe Leu 225 230 235 240 Ser Pro Arg Asp Ser Val Ala Gly Leu Thr Gly His Gly Gly His Gly 245 250 255 Thr His Thr Ala Ser Thr Ala Gly Gly Asn Asn Gly Ala Leu Ala Ser 260 265 270 Ser Asn Gly Val Ser Leu Gly Lys Ala Ser Gly Met Ala Pro Arg Ala 275 280 285 Arg Ile Ala Ala Tyr Lys Val Cys Trp Thr Ala Ala Ser Thr Gly Arg 290 295 300 Asn Gly Cys Ala Thr Ala Asp Ser Val Ala Ala Ile Asp Gln Ala Val 305 310 315 320 Lys Asp Gly Val Asn Val Ile Asn Phe Ser Ile Gly Pro Asn Ala Gly 325 330 335 Gly Gly Ala Phe Asp Glu Pro Thr Glu Val Ala Phe Leu Gly Ala Ala 340 345 350 Ala Ala Gly Val Phe Val Ala Thr Ser Gly Gly Asn Ser Gly Pro Ala 355 360 365 Thr Pro Ala Pro Asp Val Pro Ala Pro Val Ser His Ile Ser Pro Trp 370 375 380 Leu Ala Thr Val Ala Asn Ser Thr His Asn Arg Leu Tyr Ala Gly Asn 385 390 395 400 Val Ile Leu Ser Asn Gly Ala Lys Leu Glu Gly Ala Ser Ser Asn Ala 405 410 415 Lys Thr Pro Ala Leu Pro Leu Ile Arg Ser Arg Asp Ala Gly Leu Ala 420 425 430 Gly Val Ser Pro Thr Asp Val Asn Leu Leu Arg Cys Phe Gly Ala Ala 435 440 445 Asp Thr Thr Ser Ala Tyr Leu Asp Pro Ala Lys Val Ala Gly Lys Ile 450 455 460 Leu Val Cys Asp Arg Gly Gly Asn Val Leu Val Asn Lys Ser Ala Asn 465 470 475 480 Ala Lys Thr Ala Gly Ala Ala Gly Val Ile Ile Ala Asn Val Ala Gly 485 490 495 Gly Ala Asn Thr Ile Ile Asn Gln Ala His Val Leu Ser Thr Val His 500 505 510 Leu Ala Gln Ala Gln Gly Asp Ala Leu Lys Ala Phe Met Ala Val Asn 515 520 525 Pro Asp Gly Thr Ala Ala Leu Gly Glu Ile His Thr Ile Pro Asp Thr 530 535 540 Thr Val Gln Ala Pro Ile Val Ser Asp Arg Ser Ser Arg Gly Pro Asn 545 550 555 560 Val Ala Asn Ala Asn Ile Leu Lys Pro Asp Leu Ser Ala Pro Gly Thr 565 570 575 Asn Val Leu Ala Gly Val Thr Ala Asp Leu Thr Pro Ala Gln Arg Asp 580 585 590 Ala Val Ala Ala Gly Gly Val Ala Pro Val Ala Glu Trp Asp Phe Tyr 595 600 605 Ser Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Val Ala Ala Leu 610 615 620 Leu Lys Gln Gln His Pro Thr Trp Ser Pro Ala Ala Ile Lys Ser Ala 625 630 635 640 Leu Met Thr Thr Ala Phe Ser Thr Tyr Ser Asp Gly Leu Asn Gly Ser 645 650 655 Val Ser Trp Asp Ala Thr Ala Lys Asn Ser Gly Gln Leu Pro Trp Gly 660 665 670 Gln Gly Ala Gly His Ile Ala Pro Asn Ser Ala Ala Asp Pro Gly Leu 675 680 685 Val Tyr Asp Val Ser Glu Leu Asp Tyr Ala Arg Phe Leu Cys Gly Leu 690 695 700 Asn Leu Lys Val Tyr Ser Pro Ala Thr Cys Gln Ser Val Gly Thr Ile 705 710 715 720 Pro Ala Tyr Asn Leu Asn Leu Ala Ser Leu Thr Ala Ala Asn Val Leu 725 730 735 Gly Thr Gln Thr Leu Thr Arg Thr Val Thr Asn Val Gly Ala Gly Ser 740 745 750 Ala Val Tyr Asn Val Ser Ala Ser Leu Pro Gly Tyr Thr Val Ala Val 755 760 765 Thr Pro Thr Ser Leu Ser Leu Ala Pro Gly Ala Arg Ala Gln Tyr Gln 770 775 780 Val Lys Leu Thr Arg Thr Thr Ala Pro Ala Asn Thr Trp Thr Tyr Gly 785 790 795 800 Ala Leu Ser Trp Ser Asp Gly Thr His Thr Val Arg Ser Pro Leu Thr 805 810 815 Ala Arg Gly Thr Ser Leu Ala Ala Ile Pro Leu Val Ser Ser Glu Ala 820 825 830 Ala Thr Gly Ser Lys Val Leu Thr Leu Gly Thr Gly Phe Thr Gly Ala 835 840 845 Leu Val Gly Val Lys Ser Gly Leu Val Glu Ala Val Arg Glu Ala Arg 850 855 860 Thr Ile Gly Gln Ala Thr Thr Gly Ala Ala Ala Ser Ala Ala Cys Lys 865 870 875 880 Ala Gly Gly Ala Thr Gly Val Asn Val His Asn Val Val Ile Pro Ala 885 890 895 Gly Thr Leu Ala Ala Arg Phe Ala Thr Tyr Asp Gly Glu Thr Thr Gly 900 905 910 Gly Ala Asn Thr Asp Met Asp Met Glu Val Tyr Asn Ala Ala Asn Val 915 920 925 Leu Val Gly Ser Ser Gly Asn Glu Gly Ser Asn Glu Gln Val Glu Leu 930 935 940 Arg Leu Pro Ala Ala Gly Thr Tyr Lys Val Cys Val Ile Gly Tyr Ala 945 950 955 960 Pro Gln Asn Gly Gln Ala Asp Tyr Thr Leu Ser Ser Trp Val Leu Ala 965 970 975 Pro Gly Leu Ser Asn Gly Gly Phe Lys Ala Leu Met Pro Gly Thr Ala 980 985 990 Tyr Thr Gly Gly Thr Ala Thr Val Ser Leu Ser Trp Ser Asn Leu Val 995 1000 1005 Glu Gly Lys Arg Tyr Leu Gly Ala Val Gly Tyr Gln Val Gly Gly Val 1010 1015 1020 Val Gln Gly Val Thr Val Val Glu Val Asn Thr Asn Asp Pro Val Pro 1025 1030 1035 1040 Leu Phe Gln Asn Ala Arg Gly Ala Arg Pro Val Leu Ala Glu 1045 1050 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 taagcacata tgaaactacg tcccgtt 27 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer <400> 4 taagcaaagc ttttaattca gccagcaccg 30 <110> University Industry Liaison Office of Chonnam National University <120> Cold-adaptive peptidase from Janthinobacterium sp. and preparation method thereof <130> P16070971042 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 3165 <212> DNA <213> Artificial Sequence <220> <223> PAMC25641_pep DNA <400> 1 atgaaactac gtcccgtttc actcggtatc ctcttgctgg ccgccagcct ggcgcaagcg 60 gcgcaggcgc aggaacgccg ctcctacatc gtgcagctgg tcgacaagcc ggccgccacc 120 tacacgggcc aggtcgacgg cctggccgcc accaagccgg ccccgggggc acgcatcaac 180 gtgggcgccg ccgacgtgca ggcctatttg aattacctcg acaccagaca ggccgccgtg 240 gccggtaccg tcagcgcggc ggaaatcacg caccggtaca gcgtcgtctt caacggcttt 300 tccgccctgc tgacggacga tgaagtgcgg gcgctgaaga aaaacagcgg cgtggccagc 360 atcagcgccg actccatcct gcaactcgac accagctaca cgcccagctt cctgggcctg 420 gacaaaccgg gtggcatctg ggagcagctg ggcggcaagg cgcatgcggg tgaagacatc 480 atcatcggca tcgtcgacag cggcatctgg cccgagaaca cggcctttgc cgaccgcctc 540 gacgagagcg gcgtgcccag ccacagcggc aacaacgtcg tgtacggcgc gccaccggcc 600 aactggcagg gcatgtgcca gacgggcgaa ggttttggcg ccgagaattg caacaataag 660 ctgatcggtg cgcgctacta ccgcgccgcc acgtcggcgc tgcactggac ggaatttttg 720 tcgccgcgcg attccgtcgc cggcctgacg gggcacggcg gccatggcac gcacacggcg 780 tccacggccg gcggcaacaa tggcgccctg gccagctcga acggtgtgtc gctgggcaag 840 gcgtcgggca tggcgccgcg cgcccgcatc gccgcctata aagtctgctg gaccgccgcc 900 tcgacggggc gcaacggctg cgccacggcc gacagcgtgg ccgccatcga ccaggcggtc 960 aaggatggcg tcaacgtcat caacttttcc atcggcccga acgcgggcgg cggcgccttc 1020 gacgaaccga cggaagtcgc tttcctcggc gcggcggcgg ccggcgtgtt cgtcgccacg 1080 tcgggcggca attccggccc ggccacgccc gcgcccgacg tgccggcgcc cgtgtcgcac 1140 atcagcccgt ggctggcgac ggtggccaat tccacccaca accgcctgta cgcgggcaat 1200 gtgatactca gcaacggcgc caagctggaa ggcgcatcga gcaatgcgaa gacgccggcg 1260 ctgccgctga tccgctcgcg cgacgcgggc ctggccggcg tgtcgccaac ggatgtcaac 1320 ctgctgcgct gcttcggcgc ggccgatacc acctcggcct acctggaccc ggcgaaagtg 1380 gcgggcaaga tactcgtgtg cgaccgcggt ggcaacgtgc tggtcaacaa gagcgcgaat 1440 gccaaaacgg cgggcgcggc gggcgtgatc atcgccaacg tggccggtgg cgccaacacc 1500 atcatcaacc aggcgcacgt gctgtcgacc gtgcacctgg cgcaggcgca gggcgatgcg 1560 ttgaaggctt tcatggcggt caaccccgat ggcacggcgg cattgggcga aatccatacc 1620 atccccgaca cgacggtgca ggcgccgatc gtcagcgacc gttcctcgcg cgggccgaac 1680 gtggccaacg cgaacatcct gaaacccgac ctgtcggccc ccggcaccaa cgtgctggcc 1740 ggcgtgacgg ccgacctgac gccggcgcag cgcgatgccg tggcggccgg cggcgtggcg 1800 cccgtggccg agtgggattt ctattccggc acctcgatgg ccagcccgca cgtggcgggc 1860 gtggcggcat tgctcaagca gcagcacccg acctggtcgc ccgcggccat caagtcggcg 1920 ctgatgacga cggccttcag cacgtattcc gacggcttga acggttccgt gtcgtgggat 1980 gccacggcga agaactcggg ccagttgccg tggggccagg gcgccggcca catcgcgccg 2040 aacagcgcgg ccgatcccgg cctcgtgtac gacgtgtcgg aactcgacta tgcgcgcttc 2100 ctgtgcggcc tgaacctgaa ggtctacagt cccgccacct gccagtctgt cggcaccatt 2160 cccgcctaca acctgaacct ggcgtcgctg acggcggcca acgtgctggg cacgcagaca 2220 ttgacgcgca cggtgaccaa cgtgggcgcc ggcagcgccg tctacaacgt ctcggccagc 2280 ctgcccggct acacggtggc ggtgacgcca acgagtctga gcctggcgcc gggcgccagg 2340 gcgcagtacc aggtcaagct gacgcgcacc acggcgccgg ccaatacctg gacctacggc 2400 gccctgagct ggagcgatgg cacgcacacg gtgcgcagcc cgctgacggc gcgcggcacc 2460 tcgctggccg ccattccact ggtcagcagc gaagcggcca cgggcagcaa ggtgctgacc 2520 ctgggcacgg gcttcacggg cgcgctggtg ggcgtcaagt cggggctggt cgaagccgta 2580 cgtgaagcgc gcacgatcgg ccaggcgacg acgggtgctg ccgcatcggc cgcctgcaag 2640 gccggtggcg ccacgggcgt gaacgtgcac aacgtggtga tcccggccgg caccctggcg 2700 gcgcgctttg ccacctacga cggggaaacg acgggcggcg cgaataccga catggacatg 2760 gaggtctaca acgccgccaa tgtgctggtc ggcagcagcg gcaatgaagg ttccaatgag 2820 caagtggaac tgcgcctgcc cgccgccggc acgtacaagg tgtgcgtgat cggctacgcg 2880 ccgcaaaacg gccaggccga ctacactttg tcgtcgtggg tgctggcgcc gggcctgtcg 2940 aatggcggct tcaaggcgtt gatgccgggc accgcctaca cgggcggcac ggcgacggtc 3000 tcgctgagct ggtccaacct ggtcgaaggc aagcgctacc tgggcgccgt cggctaccaa 3060 gtgggcggcg tggtgcaggg cgtgacggtg gtcgaggtca ataccaatga ccccgtgccg 3120 ctgttccaga acgcgcgcgg cgccaggccg gtgctggctg aataa 3165 <210> 2 <211> 1054 <212> PRT <213> Artificial Sequence <220> <223> PAMC25641_pep Protein <400> 2 Met Lys Leu Arg Pro Val Ser Leu Gly Ile Leu Leu Leu Ala Ala Ser 1 5 10 15 Leu Ala Gln Ala Gln Ala Gln Glu Arg Arg Ser Tyr Ile Val Gln 20 25 30 Leu Val Asp Lys Pro Ala Ala Thr Tyr Thr Gly Gln Val Asp Gly Leu 35 40 45 Ala Ala Thr Lys Pro Ala Pro Gly Ala Arg Ile Asn Val Gly Ala Ala 50 55 60 Asp Val Gln Ala Tyr Leu Asn Tyr Leu Asp Thr Arg Gln Ala Ala Val 65 70 75 80 Ala Gly Thr Val Ser Ala Ala Glu Ile Thr His Arg Tyr Ser Val Val 85 90 95 Phe Asn Gly Phe Ser Ala Leu Leu Thr Asp Asp Glu Val Arg Ala Leu 100 105 110 Lys Lys Asn Ser Gly Val Ala Ser Ile Ser Ala Asp Ser Ile Leu Gln 115 120 125 Leu Asp Thr Ser Tyr Thr Pro Ser Phe Leu Gly Leu Asp Lys Pro Gly 130 135 140 Gly Ile Trp Glu Gln Leu Gly Gly Lys Ala His Ala Gly Glu Asp Ile 145 150 155 160 Ile Ile Gly Ile Val Asp Ser Gly Ile Trp Pro Glu Asn Thr Ala Phe 165 170 175 Ala Asp Arg Leu Asp Glu Ser Gly Val Ser Ser His Ser Gly Asn Asn 180 185 190 Val Val Tyr Gly Ala Pro Pro Ala Asn Trp Gln Gly Met Cys Gln Thr 195 200 205 Gly Glu Gly Phe Gly Ala Glu Asn Cys Asn Asn Lys Leu Ile Gly Ala 210 215 220 Arg Tyr Tyr Arg Ala Ala Thr Ser Ala Leu His Trp Thr Glu Phe Leu 225 230 235 240 Ser Pro Arg Asp Ser Val Ala Gly Leu Thr Gly His Gly Gly His Gly 245 250 255 Thr His Thr Ala Ser Thr Ala Gly Gly Asn Asn Gly Ala Leu Ala Ser 260 265 270 Ser Asn Gly Val Ser Leu Gly Lys Ala Ser Gly Met Ala Pro Arg Ala 275 280 285 Arg Ile Ala Ala Tyr Lys Val Cys Trp Thr Ala Ala Ser Thr Gly Arg 290 295 300 Asn Gly Cys Ala Thr Ala Asp Ser Val Ala Ala Ile Asp Gln Ala Val 305 310 315 320 Lys Asp Gly Val Asn Val Ile Asn Phe Ser Ile Gly Pro Asn Ala Gly 325 330 335 Gly Gly Ala Phe Asp Glu Pro Thr Glu Ala Phe Leu Gly Ala Ala 340 345 350 Ala Ala Gly Val Ala Val Ala Thr Ser Gly Gly Asn Ser Gly Pro Ala 355 360 365 Thr Pro Ala Pro Asp Val Pro Ala Pro Val Ser His Ile Ser Pro Trp 370 375 380 Leu Ala Thr Val Ala Asn Ser Thr His Asn Arg Leu Tyr Ala Gly Asn 385 390 395 400 Val Ile Leu Ser Asn Gly Ala Lys Leu Glu Gly Ala Ser Ser Asn Ala 405 410 415 Lys Thr Pro Ala Leu Pro Leu Ile Arg Ser Ser Asp Ala Gly Leu Ala 420 425 430 Gly Val Ser Pro Thr Asp Val Asn Leu Leu Arg Cys Phe Gly Ala Ala 435 440 445 Asp Thr Thr Ser Ala Tyr Leu Asp Pro Ala Lys Val Ala Gly Lys Ile 450 455 460 Leu Val Cys Asp Arg Gly Gly Asn Val Leu Val Asn Lys Ser Ala Asn 465 470 475 480 Ala Lys Thr Ala Gly Ala Ala Gly Ala Asn Val Ala Gly 485 490 495 Gly Ala Asn Thr Ile Ile Asn Gln Ala His Val Leu Ser Thr Val His 500 505 510 Leu Ala Gln Ala Gln Gly Asp Ala Leu Lys Ala Phe Met Ala Val Asn 515 520 525 Pro Asp Gly Thr Ala Ala Leu Gly Glu Ile His Thr Ile Pro Asp Thr 530 535 540 Thr Val Gln Ala Pro Ile Val Ser Asp Arg Ser Ser Arg Gly Pro Asn 545 550 555 560 Val Ala Asn Ala Asn Ile Leu Lys Pro Asp Leu Ser Ala Pro Gly Thr 565 570 575 Asn Val Leu Ala Gly Val Thr Ala Asp Leu Thr Pro Ala Gln Arg Asp 580 585 590 Ala Val Ala Gly Aly Gly Val Ala Pro Ala Glu Trp Asp Phe Tyr 595 600 605 Ser Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Val Ala Ala Leu 610 615 620 Leu Lys Gln Gln His Pro Thr Trp Ser Pro Ala Ala Ile Lys Ser Ala 625 630 635 640 Leu Met Thr Thr Ala Phe Ser Thr Tyr Ser Asp Gly Leu Asn Gly Ser 645 650 655 Val Ser Trp Asp Ala Thr Ala Lys Asn Ser Gly Gln Leu Pro Trp Gly 660 665 670 Gln Gly Ala Gly His Ile Ala Pro Asn Ser Ala Ala Asp Pro Gly Leu 675 680 685 Val Tyr Asp Val Ser Glu Leu Asp Tyr Ala Arg Phe Leu Cys Gly Leu 690 695 700 Asn Leu Lys Val Tyr Ser Pro Ala Thr Cys Gln Ser Val Gly Thr Ile 705 710 715 720 Pro Ala Tyr Asn Leu Asn Leu Ala Ser Leu Thr Ala Ala Asn Val Leu 725 730 735 Gly Thr Gln Thr Leu Thr Arg Thr Val Thr Asn Val Gly Ala Gly Ser 740 745 750 Ala Val Tyr Asn Val Ser Ala Ser Leu Pro Gly Tyr Thr Val Ala Val 755 760 765 Thr Pro Thr Ser Leu Ser Leu Ala Pro Gly Ala Arg Ala Gln Tyr Gln 770 775 780 Val Lys Leu Thr Arg Thr Thr Ala Pro Ala Asn Thr Trp Thr Tyr Gly 785 790 795 800 Ala Leu Ser Trp Ser Asp Gly Thr His Thr Val Arg Ser Ser Leu Thr 805 810 815 Ala Arg Gly Thr Ser Leu Ala Ile Pro Leu Val Ser Ser Glu Ala 820 825 830 Ala Thr Gly Ser Lys Val Leu Thr Leu Gly Thr Gly Phe Thr Gly Ala 835 840 845 Leu Val Gly Val Lys Ser Gly Leu Val Glu Ala Val Arg Glu Ala Arg 850 855 860 Thr Ile Gly Gln Ala Thr Thr Gly Ala Ala Ala Ser Ala Ala Cys Lys 865 870 875 880 Ala Gly Aly Thr Gly Aly Gly Aly Gly Aly Gly Aly 885 890 895 Gly Thr Leu Ala Ala Arg Phe Ala Thr Tyr Asp Gly Glu Thr Thr Gly 900 905 910 Gly Ala Asn Thr Asp Met Asp Met Glu Val Tyr Asn Ala Ala Asn Val 915 920 925 Leu Val Gly Ser Ser Gly Asn Glu Gly Ser Asn Glu Gln Val Glu Leu 930 935 940 Arg Leu Pro Ala Ala Gly Thr Tyr Lys Val Cys Val Ile Gly Tyr Ala 945 950 955 960 Pro Gln Asn Gly Gln Ala Asp Tyr Thr Leu Ser Ser Trp Val Leu Ala 965 970 975 Pro Gly Leu Ser Asn Gly Gly Phe Lys Ala Leu Met Pro Gly Thr Ala 980 985 990 Tyr Thr Gly Gly Thr Ala Thr Val Ser Leu Ser Trp Ser Asn Leu Val 995 1000 1005 Glu Gly Lys Arg Tyr Leu Gly Ala Val Gly Tyr Gln Val Gly Gly Val 1010 1015 1020 Val Gln Gly Val Thr Val Val Glu Val Asn Thr Asn Asp Pro Val Pro 1025 1030 1035 1040 Leu Phe Gln Asn Ala Arg Gly Ala Arg Pro Val Leu Ala Glu 1045 1050 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward primer <400> 3 taagcacata tgaaactacg tcccgtt 27 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse Primer <400> 4 taagcaaagc ttttaattca gccagcaccg 30

Claims

Peptidase derived from Janthinobacterium sp. PAMC 25641 having the amino acid sequence of SEQ ID NO: 1.

A nucleic acid molecule encoding the peptidase of claim 1.

3. The method of claim 2,
Wherein the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 2.

A vector comprising the nucleic acid molecule of claim 2 or 3.

A transformant, except human, transformed with the vector of claim 4.

6. The method of claim 5,
Wherein the transformant is a bacterium, a cyanobacterium or a microalgae.

A recombinant vector pET-28a (+) having the cleaved map of FIG. 5 inserted with a peptidase gene comprising the nucleic acid molecule of claim 2 or 3: PAMC25641_pep.