KR100310932B1 - Discovery of Staphylococcus haemolyticus L62(KCTC 8957P) producing a novel lipase and development of its efficient production method using Escherichia coli BL21(DE3)/pSHML(KCTC 8956P) - Google Patents
Discovery of Staphylococcus haemolyticus L62(KCTC 8957P) producing a novel lipase and development of its efficient production method using Escherichia coli BL21(DE3)/pSHML(KCTC 8956P) Download PDFInfo
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- KR100310932B1 KR100310932B1 KR1019990044831A KR19990044831A KR100310932B1 KR 100310932 B1 KR100310932 B1 KR 100310932B1 KR 1019990044831 A KR1019990044831 A KR 1019990044831A KR 19990044831 A KR19990044831 A KR 19990044831A KR 100310932 B1 KR100310932 B1 KR 100310932B1
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- lipase
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- pshml
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- 102000004882 Lipase Human genes 0.000 title claims abstract description 122
- 239000004367 Lipase Substances 0.000 title claims abstract description 122
- 235000019421 lipase Nutrition 0.000 title claims abstract description 122
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 title claims abstract description 18
- 241000191984 Staphylococcus haemolyticus Species 0.000 title claims abstract description 18
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- C12P7/00—Preparation of oxygen-containing organic compounds
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Abstract
본 발명은 토양으로부터 분리된 것으로 저온성 리파제활성능이 우수한 신균주 스타필로코커스 헤모리티커스(Staphylococcus haemolyticus) L62(KCTC 8957P) 및 이러한 신균주로부터 생산된 것으로 비이온성 계면활성제에 의해 활성이 크게 증가하고 저온에서도 높은 활성을 가지는 L62 리파제 그리고, T7 프로모터를 이용하여 상기한 L62 리파제를 대량생산하는 형질전환된 대장균 BL21(DE3)/pSHML(KCTC 8956P) 및 리소스(Resource) Q 컬럼을 이용한 L62 리파제의 정제방법에 관한 것이다.The present invention is isolated from the soil and has a high temperature low temperature lipase activity, Staphylococcus haemolyticus L62 (KCTC 8957P) and produced from these new strains, and is highly active by a nonionic surfactant L62 lipase with increased and high activity even at low temperatures, and L62 lipase using transformed Escherichia coli BL21 (DE3) / pSHML (KCTC 8956P) and Resource Q columns to mass produce L62 lipase using the T7 promoter. It relates to a purification method of.
Description
본 발명은 토양으로부터 분리된 것으로 저온성 리파제활성능이 우수한 신균주 스타필로코커스 헤모리티커스(Staphylococcus haemolyticus) L62(KCTC 8957P) 및 이러한 신균주로부터 생산된 것으로 비이온성 계면활성제에 의해 활성이 크게 증가하고 저온에서도 높은 활성을 가지는 L62 리파제 그리고, T7 프로모터를 이용하여 상기한 L62 리파제를 대량생산하는 형질전환된 대장균 BL21(DE3)/pSHML(KCTC 8956P) 및 리소스(Resource) Q 컬럼을 이용한 L62 리파제의 정제방법에 관한 것이다.The present invention is isolated from the soil and has a high temperature low temperature lipase activity, Staphylococcus haemolyticus L62 (KCTC 8957P) and produced from these new strains, and is highly active by a nonionic surfactant L62 lipase with increased and high activity even at low temperatures, and L62 lipase using transformed Escherichia coli BL21 (DE3) / pSHML (KCTC 8956P) and Resource Q columns to mass produce L62 lipase using the T7 promoter. It relates to a purification method of.
효소는 생체내의 각종 생화학 반응을 촉진시키는 촉매로서 널리 연구되어 왔다. 효소는 반응이 상온·상압에서 일어나고 고효율성 및 특이성 등의 장점에 기인하여 여러 가지 생물전환반응의 효소촉매로 이용되고 있으며, 기존의 화학합성법을 대체함으로써 점차 산업적 응용범위가 확대되는 중요한 분야이다.Enzymes have been widely studied as catalysts for promoting various biochemical reactions in vivo. Enzymes are used as enzyme catalysts for various bioconversion reactions due to the advantages of high efficiency and specificity, and reactions at room temperature and atmospheric pressure, and are an important field in which industrial applications are gradually expanded by replacing existing chemical synthesis methods.
또한, 에스테르화합물이 식품, 화학, 제약 및 환경소재 공업에 이용도가 높은 정밀화학제품에 많이 포함되어 있기 때문에, 에스테르결합의 합성관련 효소는 생물촉매분야의 40% 이상을 점유하는 매우 중요한 효소이다.In addition, since ester compounds are frequently included in fine chemicals, which are widely used in the food, chemical, pharmaceutical, and environmental materials industries, the synthesis-related enzymes of ester bonds are very important enzymes occupying more than 40% of the biocatalyst field. .
기존의 화학적인 방법으로 에스테르화합물을 합성하는 경우, 700 psi의 고압 및 250℃ 이상의 고온에서 합성되기 때문에 에너지가 많이 소모되며 품질에 나쁜 영향을 주는 여러 가지 부반응이 일어날 뿐 아니라, 전환율 및 광학순도가 낮아서 고 순도의 정밀화학제품 생산에 어려움이 있어 왔다.When the ester compound is synthesized by the conventional chemical method, since it is synthesized at a high pressure of 700 psi and a high temperature of 250 ° C. or higher, energy consumption and various side reactions that adversely affect the quality occur, as well as conversion and optical purity. It has been difficult to produce high-purity fine chemicals.
따라서, 최근에 상기의 단점을 없애기 위해 이상계, 미수계, 비수계 및 역상계에서의 리파제를 이용한 효소촉매 반응계가 개발됨에 따라, 고부가가치의 정밀화학제품의 제조에 여러 가지 리파제가 효소촉매로 이용되기 시작하였다. 즉, 장시간의 효소 반응 공정에서 그 활성을 잃지 않으며, 저온, 고온, 알칼리, 유기용매 및 계면활성제 처리 등의 열악한 반응 환경에서도 큰 활성을 보이는 리파제의 탐색과 개발이 활발히 진행되고 있다. 특히, 세제첨가제로 사용되거나 고온에서 불안정한 물질의 합성을 위해서는 저온성 및 계면 활성제에 대한 내성 등의 특성을 갖는 리파제가 요구된다.Therefore, in recent years, in order to eliminate the above-mentioned disadvantages, enzyme catalyst reaction systems using lipases in ideal, non-aqueous, non-aqueous and reverse phase systems have been developed. It started to be. That is, the search and development of lipases, which do not lose their activity in a long time enzymatic reaction process and show great activity even in poor reaction environments such as low temperature, high temperature, alkali, organic solvent and surfactant treatment, are actively progressing. In particular, for the synthesis of materials used as detergent additives or unstable at high temperatures, lipases having properties such as low temperature and resistance to surfactants are required.
그러나, 현재까지 개발된 대부분 리파제의 경우, 저온(4℃)에서 효소의 활성이 매우 약하며, 비이온성 계면활성제에 의해 효소의 활성이 대부분 없어진다.However, most of the lipases developed to date, the enzyme activity is very weak at low temperatures (4 ℃), the activity of the enzyme is largely lost by the nonionic surfactant.
이에, 본 발명자들은 계면활성제에 의해 활성화되고, 저온에서도 효소의 활성이 우수한 리파제를 생산하는 균주를 탐색하고, 이러한 리파제를 대량생산하는 시스템을 개발함으로써, 본 발명을 완성하였다.Thus, the present inventors completed the present invention by searching for a strain that is activated by a surfactant and produces a lipase excellent in enzyme activity even at low temperatures, and develops a system for mass production of such lipase.
따라서, 본 발명은 비이온성 계면활성제에 의해 리파제의 활성이 안정 및 증가되는 특성과 상온에서 불안정한 고가의 불포화 지방산의 에스테르화합물의 합성을 위해 저온에서 활성이 높으며 1,3-위치 특이성을 갖는 신규 리파제를 대량으로 제공하는데 그 목적이 있다.Therefore, the present invention is a novel lipase having high activity at low temperature and having 1,3-position specificity for the synthesis of ester compound of expensive unsaturated fatty acid which is stable and increased activity of lipase by nonionic surfactant and unstable at room temperature. The purpose is to provide a large amount.
도 1은 온도에 따른 L62 리파제의 가수분해활성과 칼슘 또는 EDTA의 존재하에서 효소를 30분간 방치한 후의 L62 리파제의 잔존활성을 나타내는 그래프이다.1 is a graph showing the hydrolytic activity of L62 lipase with temperature and the residual activity of L62 lipase after the enzyme was left for 30 minutes in the presence of calcium or EDTA.
도 2는 온도에 따른 L62 리파제의 활성화 에너지와 불활성화 에너지를 나타내는 그래프이다.Figure 2 is a graph showing the activation energy and inactivation energy of L62 lipase with temperature.
도 3은 머추어(mature) 리파아제가 삽입된 재조합 플라스미드 pBSII SK의 모식도이다.Figure 3 is a schematic diagram of recombinant plasmid pBSII SK inserted with mature lipase.
도 4는 L62 리파제의 대량발현 벡터인 pSHML의 제조과정을 보여주는 모식도이다.Figure 4 is a schematic diagram showing the manufacturing process of pSHML, a mass expression vector of L62 lipase.
도 5a는 pSHML벡터로 형질전환된 대장균 BL21(DE3)/pSHML으로부터 L62 리파제가 대량 발현됨을 SDS-PAGE로 확인한 사진이고, 도 5b는 도 5a의 결과를 나타내는 그래프이다.Figure 5a is a photograph confirming the mass expression of L62 lipase from Escherichia coli BL21 (DE3) / pSHML transformed with a pSHML vector by SDS-PAGE, Figure 5b is a graph showing the result of Figure 5a.
도 6은 형질전환된 대장균 BL21(DE3)/pSHML(KCTC 8956P)로부터 대량생산된 L62 리파제가 리소스(Resource) Q 컬럼을 통해 순수 분리됨을 나타내는 사진이다.6 is a photograph showing that L62 lipase mass-produced from transformed Escherichia coli BL21 (DE3) / pSHML (KCTC 8956P) is purely separated through a Resource Q column.
도 7은 L62 리파제가 트리올레인을 1,3-위치 특이적으로 가수분해함을 보여주는 사진이다.7 is a photograph showing that L62 lipase specifically hydrolyzes triolein at 1,3-position.
도 8은 트윈 80이 처리된 L62 리파제 및 무처리된 L62 리파제에 의한 여러 가지 천연 지방질의 가수분해 활성을 나타내는 그래프이다.8 is a graph showing the hydrolytic activity of various natural fats by T62 80 treated L62 lipase and untreated L62 lipase.
본 발명은 신균주 스타필로코커스 헤모리티커스(Staphylococcus haemolyticus) L62(KCTC 8957P) 및 이로부터 생산되고 비이온성 계면활성제에 의해 활성이 증가되는 저온성 L62 리파제를 특징으로 한다.The present invention features the new strain Staphylococcus haemolyticus L62 (KCTC 8957P) and low temperature L62 lipase produced therefrom and increased in activity by a nonionic surfactant.
또한, 본 발명은 L62 리파제의 대량생산을 위해 형질전환된 대장균 BL21(DE3)/pSHML(KCTC 8956P) 및 이로부터 생산된 리파제를 정제하는 방법을 특징으로 한다.The invention also features a method for purifying transformed E. coli BL21 (DE3) / pSHML (KCTC 8956P) and lipases produced therefrom for mass production of L62 lipases.
이와 같은 본 발명을 상세히 설명하면 다음과 같다.The present invention will be described in detail as follows.
본 발명에서는 리파제 생산균주를 토양 샘플에서 분리한 후, 이를 액체 배양하여 배양 상등액의 리파제 활성이 우수한 균주를 최종적으로 선별하여, 선별된 균주의 형태학적 및 생화학적 특성 조사를 통해 균주 동정을 실시하였다[Bergey'sManual of Determinative Bacteriology].In the present invention, the lipase producing strain was isolated from the soil sample, and then liquid cultured to finally select strains excellent in lipase activity of the culture supernatant, and strain identification was carried out by examining the morphological and biochemical properties of the selected strains. Bergy's Manual of Determinative Bacteriology.
상기 신균주는 형태학적으로 운동성이 없고 그람양성 구균형태이다. 또한, 신균주는 45℃에서 성장가능하고, 탄소원으로 글루코스, 슈크로스 및 트리할로스(trehalose)를 이용하였다. 또한, 신균주는 알기닌 다이하이드롤라제(arginine dihydrolase)의 활성은 있지만, 우레아제(urease), 베타 β-갈락토시다제 및 β-글루코시다제의 활성이 없는 것으로 밝혀졌다.The new strain is morphologically nonmotile and is in Gram-positive cocci form. In addition, the new strain was capable of growing at 45 ° C., and glucose, sucrose and trihalose were used as carbon sources. In addition, the new strain was found to have the activity of arginine dihydrolase but no activity of urease, beta β-galactosidase and β-glucosidase.
상기한 바와 같이, 신균주의 형태학적 및 생화학적 특성을 살펴본 바에 의하면, 본 발명에 따른 신균주는 스타필로코커스 헤모리티커스(Staphylococcus haemolyticus)의 전형적인 특성을 가진다.As described above, according to the morphological and biochemical properties of the new strain, the new strain according to the present invention has typical characteristics of Staphylococcus haemolyticus .
한편, 상기 신균주로부터 생산되는 리파제를 암호화하는 약 4.2 kb의 DNA를 클로닝하고, 이러한 DNA로부터 아미노산 서열을 분석한 결과, 이는 60개의 아미노산으로 이루어진 신호서열(signal sequence), 259개의 아미노산으로 이루어진 프로펩타이드(propeptide) 및 392개의 아미노산으로 이루어진 머추어(mature) 리파제로 구성된 프리프로리파제(preprolipase) 형태로 생산되는 것을 확인하였으며, 기존의 스타필로코커스 속의 다른 균주에 의해 생산되는 리파제와 아미노산 상동성이 67%이하인 신규 리파제임을 확인하였다.On the other hand, cloning the DNA of about 4.2 kb encoding the lipase produced from the new strain, and analyzed the amino acid sequence from this DNA, which is a signal sequence consisting of 60 amino acids, a pro consisting of 259 amino acids It was confirmed that it is produced in the form of a preprolipase consisting of a peptide (mature lipase) consisting of peptide and 392 amino acids, and amino acid homology with lipase produced by other strains of the genus Staphylococcus It was confirmed that the lipase is less than 67%.
이에, 상기 신규 리파제를 생산하는 신균주를 스타필로코커스 헤모리티커스(Staphylococcus haemolyticus; 이하 'S. haemolyticus'로 약함) L62로 명명하고, 이를 생명공학연구소 유전자은행에 1999년 8월 18일자로 기탁하여 수탁번호 KCTC 8957P를 부여받았다.Accordingly, the novel strain producing the new lipase was named Staphylococcus haemolyticus (hereinafter abbreviated as ' S. haemolyticus ') L62, which was assigned to the Biotechnology Research Institute Gene Bank on August 18, 1999. The deposit was given accession number KCTC 8957P.
또한, 상기 신균주 스타필로코커스 헤모리티커스(S. haemolyticus) L62에 의해 생산되는 리파제(이하 'L62 리파제'로 약함)의 특성을 조사한 결과, 효소의 활성은 55℃까지 안정하며, 바람직한 가수분해 활성 온도는 28℃이다. 또한, L62 리파제의 활성은 pH 5∼11.5의 범위에서 안정하며, 바람직한 pH는 8.5이다.In addition, as a result of examining the properties of the lipase produced by the strain S. haemolyticus L62 (hereinafter referred to as 'L62 lipase'), the activity of the enzyme is stable up to 55 ° C., and the preferred valence The decomposition activity temperature is 28 ° C. Moreover, the activity of L62 lipase is stable in the range of pH 5-11.5, and preferable pH is 8.5.
본 발명의 L62 리파제는 트리올레인의 1,3-위치를 특이적으로 분해하는 1,3-위치 특이성을 갖고 있을 뿐만 아니라, 저온(4℃)에서도 높은 활성을 가진다. 또한, L62 리파제는 대부분의 비이온성 계면활성제에 대해 안정하였으며, 오히려 이러한 계면활성제에 의해 효소의 활성이 증가되었을 뿐만 아니라, 효소의 기질특이성, 최적 작용 pH와 온도 및 분자량이 변화되는 현상을 확인하였다.L62 lipase of the present invention not only has 1,3-position specificity to specifically decompose the 1,3-position of triolein, but also has high activity even at low temperature (4 ° C). In addition, L62 lipase was stable to most nonionic surfactants, and rather, the activity of enzymes was increased by these surfactants, and the substrate specificity, optimal pH, temperature and molecular weight of the enzymes were confirmed. .
예를 들면, 트윈 80을 L62 리파제에 처리한 경우, 대부분의 기질에 대한 L62 리파제의 가수분해 활성이 증가하였으며, 특히 불포화 지방산이 함유된 기질에 대한 활성이 10배 가량 증가하였다. 또한, 리파제의 바람직한 pH가 원래 8.5인데 비해서, 트윈 80이 처리된 리파제는 pH가 7.5∼10인 넓은 범위에서 최적 작용을 하였다.For example, when Tween 80 was treated with L62 lipase, the hydrolytic activity of L62 lipase on most substrates was increased, particularly about 10-fold increase in activity on substrates containing unsaturated fatty acids. In addition, the preferred pH of the lipase was 8.5, whereas the lipase treated with Tween 80 performed optimally over a wide range of pH 7.5-10.
따라서, L62 리파아제의 작용 온도 및 pH를 용도에 따라 조절할 수 있기 때문에 다양한 반응 조건에서 유용한 지방산을 생산할 수 있는 장점을 가지며, 공정 과정 동안 L62 리파아제가 계면활성제에 의해서 불활성화되지 않기 때문에 매우 유용하다.Therefore, since the operating temperature and pH of L62 lipase can be adjusted according to the use, it has the advantage of producing useful fatty acids under various reaction conditions, and is very useful because L62 lipase is not inactivated by a surfactant during the process.
예를 들면, L62 리파아제는 고온에서 불안정한 불포화 지방산이 다량 함유된 편유(linseed oil) 및 맥아유(wheat germ oil)에 대한 높은 효소활성을 가지므로,트랜스 에스테르화 반응을 이용한 고가의 고급유지의 개발에 유용하게 사용될 수 있다.For example, L62 lipase has high enzymatic activity against linseed and malt oils containing large amounts of unsaturated fatty acids that are unstable at high temperatures, and therefore, development of expensive high-quality fats and oils using trans esterification reactions. It can be usefully used.
한편, L62 리파제의 산업적 이용을 위해 리파제 DNA를 포함하는 대량생산용 재조합 플라스미드(pSHML)를 제조한 후, 이러한 플라스미드로 형질전환된 대장균 BL21(DE3)/pSHML을 제조하여, 이를 생명공학연구소 유전자은행에 1999년 8월 18일자로 기탁하여 수탁번호 KCTC 8956P를 부여받았다.On the other hand, for the industrial use of L62 lipase, a recombinant plasmid for mass production containing lipase DNA (pSHML) was prepared, and E. coli BL21 (DE3) / pSHML transformed with the plasmid was prepared, which was then used by the Institute of Biotechnology. Was deposited on August 18, 1999 and received accession number KCTC 8956P.
상기 형질전환된 대장균 BL21(DE3)/pSHML을 이용하여 L62 리파제의 고발현 실험을 수행한 결과, 원균주인 스타필로코커스 헤모리티커스(S. haemolyticus) L62 보다 L62 리파제의 생산량이 8.2배 증가되고, 활성 리파제가 전체 단백질의 약 30 중량%인 80,000 U/ℓ까지 생산되었다. 다른 대부분의 균주에 의해 생산된 리파제가 효소의 활성이 없는 불용성 단백질로 생산되는데 반해, L62 리파제의 대부분은 활성을 보이는 단백질로 생산되었다.As a result of the high expression experiment of L62 lipase using the transformed Escherichia coli BL21 (DE3) / pSHML, the production of L62 lipase was 8.2 times higher than that of S. haemolyticus L62. , Active lipase was produced up to 80,000 U / l, about 30% by weight of total protein. Lipases produced by most other strains were produced as insoluble proteins without enzyme activity, whereas most of L62 lipases were produced as proteins that showed activity.
또한, 본 발명은 형질전환된 대장균 BL21(DE3)/pSHML으로부터 대량생산된 리파제의 효과적인 분리정제법을 확립하였다. 즉, 높은 등전점을 갖는 L62 리파제의 특성을 이용하여 음이온 교환수지인 리소스(Resourse) Q 컬럼을 통해 정제분리시켰다. 정제된 L62 리파제는 비활성이 600 U/mg이고, 약 40%의 회수율을 보였다.In addition, the present invention has established an effective isolation and purification method of mass produced lipase from transformed Escherichia coli BL21 (DE3) / pSHML. In other words, the L62 lipase having a high isoelectric point was used for purification through a Resource Q column, which is an anion exchange resin. Purified L62 lipase had a specific activity of 600 U / mg and showed a recovery of about 40%.
현재 에스테르 관련 효소촉매인 리파제 및 에스터라제의 경우, 화학촉매에 비해서 가격이 높기 때문에 일부 고부가가치의 물질전환을 제외하고는 사용하기 어려운 실정을 고려하면, 상기의 형질전환된 대장균주를 이용한 대량발현 시스템의리파제의 고발현을 통하여 효소를 대량생산함으로써 가격 경쟁력을 확보할 수 있게 되었다.In the case of lipases and esterases, which are enzyme enzymes currently used, they are more expensive than chemical catalysts, and thus are difficult to use except for some high value-added substance conversion. The high expression of the lipase in the expression system allows mass production of enzymes to secure price competitiveness.
이하, 비이온성 계면활성제에 의해 활성화되는 저온성 L62 리파제를 생산하는 신균주 스타필로코커스 헤모리티커스(S. haemolyticus) L62, L62 리파제를 암호화하는 유전자, 이러한 유전자를 포함하는 재조합 플라스미드로 형질전환된 대장균 BL21(DE3)/pSHML 및 대량생산된 L62 리파제의 정제방법에 관하여 다음의 실시예를 통하여 구체적으로 설명하는 바, 본 발명이 이들에 한정되는 것은 아니다.Hereinafter, transformed with the strain S. haemolyticus L62, a gene encoding L62 lipase, a recombinant plasmid containing such a gene, which produces a low temperature L62 lipase activated by a nonionic surfactant. The method for purifying the isolated E. coli BL21 (DE3) / pSHML and the mass-produced L62 lipase will be described in detail through the following examples, but the present invention is not limited thereto.
실시예 1: L62 리파제의 생산 및 특성Example 1 Production and Properties of L62 Lipase
신균주 스타필로코커스 헤모리티커스(S. haemolyticus) L62을 LB배지(1% 트립톤, 0.5% 효모추출액 및 0.5% 소금)를 사용하여 37℃에서 20시간 배양한 후, 배양 상등액에서 L62 리파제 9,800 U/ℓ를 얻었다. 이러한 결과는 다른 균주로부터 생산되는 리파제 3,000 U/ℓ의 약 3배에 해당되는 양이다.The strain S. haemolyticus L62 was incubated at 37 ° C. for 20 hours using LB medium (1% tryptone, 0.5% yeast extract and 0.5% salt), and then L62 lipase in the culture supernatant. 9,800 U / L was obtained. This result is about three times the amount of 3,000 U / l lipase produced from other strains.
1.2ℓ의 배양액을 원심 분리하여 균체를 제거한 후, 황산암모늄 침전, DEAE와 CM-세파로스(Sepharose) CL-6B 컬럼 및 리소스(Resource) S 컬럼을 사용하여 L62 리파제를 정제분리하고, 다음과 같이 L62 리파제의 특성을 조사하였다.After centrifugation of 1.2 L of the culture solution to remove the cells, L62 lipase was purified and purified using ammonium sulfate precipitation, DEAE and CM-Sepharose CL-6B columns, and Resource S columns. The properties of L62 lipase were investigated.
1) L62 리파제의 분자량 및 N-말단 아미노산 서열 분석1) Molecular weight and N-terminal amino acid sequence analysis of L62 lipase
L62 리파제의 분자량을 검토하기 위하여 12% SDS-PAGE를 수행한 결과, 분자량 45,000 위치의 단일 밴드로 나타났다. 이러한 L62 리파제의 단백질 밴드를겔상에서 추출한 후, N-말단의 아미노산을 분석한 결과, 표 1과 같은 아미노산 서열로 나타났는데, 스타필로코커스 속의 다른 균주에 의해 생산되는 리파제와 상당히 다른 신규 효소임을 알 수 있다.A 12% SDS-PAGE was performed to examine the molecular weight of L62 lipase, resulting in a single band of 45,000 molecular weight positions. After extracting the protein band of the L62 lipase on the gel, and analyzed the amino acid of the N-terminal, the amino acid sequence shown in Table 1, it shows a novel enzyme significantly different from the lipase produced by other strains of Staphylococcus Can be.
2) L62 리파제의 활성2) Activity of L62 Lipase
L62 리파제 활성에 대한 pH의 영향을 조사한 결과, pH 8.5에서 최대의 활성을 나타내었고, 정제된 효소의 pH 안정성을 조사한 결과, pH 5.0∼11.5 범위에서 안정한 효소 활성을 보였다.As a result of investigating the effect of pH on L62 lipase activity, it showed the maximum activity at pH 8.5, and the stability of the purified enzyme showed a stable enzyme activity in the pH range of 5.0 to 11.5.
한편, L62 리파제 활성에 미치는 온도의 영향을 조사한 결과, 도 1에 나타낸 바와 같이, 28℃에서 최적활성을 보였고, 4℃에서도 최대활성의 30%의 활성을 보이는 것으로 나타났다.On the other hand, as a result of examining the effect of temperature on L62 lipase activity, as shown in FIG.
L62 리파제의 활성화 에너지는 도 2에 나타낸 바와 같이 8.63 kcal/mol로 저온성 효소의 전형적인 특성을 나타낸다. 또한, L62 리파제의 열에 대한 안정성을 검토한 결과, 50℃에서 30분 동안 안정되었고, 이것으로 L62 리파제는 비교적고온에서도 안정한 효소임을 알 수 있다.The activation energy of L62 lipase is 8.63 kcal / mol, which is typical of the low temperature enzyme as shown in FIG. In addition, as a result of examining the heat stability of L62 lipase, it was stable at 50 ° C. for 30 minutes, which shows that L62 lipase is a stable enzyme even at a relatively high temperature.
실시예 2: L62 리파제 DNA의 클로닝 및 아미노산 서열결정Example 2: Cloning and Amino Acid Sequencing of L62 Lipase DNA
스타필로코커스 해모리티커스(S. haemolyticus) L62의 DNA를 제한효소ClaI 및EcoRV로 완전히 자른 후, 동일한 제한효소로 자른 플라스미드 pBluescriptII SK와 연결(ligation)하고 대장균 XL1 Blue로 형질전환을 수행하여 리파제 기질이 함유된 배지에서 활성을 보이는 5개의 콜로니(colony)을 얻었다.DNA of Staphylococcus haemolyticus L62 was completely cut with restriction enzymes Cla I and Eco RV, followed by ligation with plasmid pBluescriptII SK cut with the same restriction enzyme and transformation with Escherichia coli XL1 Blue. Five colonies were obtained which showed activity in the medium containing the lipase substrate.
상기 콜로니로부터 플라스미드 DNA를 분리한 결과, 모두 2.9 kb 벡터 DNA에 4.2 kb의 외래 DNA가 삽입되어 있는 것으로 나타났다. 이러한 4.2 kb의 외래 DNA를 갖는 재조합 플라스미드를 pSHL로 명명하였다. 상기 콜로니의 L62 리파제 생산능은 2,500 U/ℓ이므로 4.2 kb 단편 안에 완전한 리파제 유전자가 존재함을 확인할 수 있었다.Plasmid DNA was isolated from the colonies, and all of them showed that 4.2 kb of foreign DNA was inserted into the 2.9 kb vector DNA. This recombinant plasmid with this 4.2 kb of foreign DNA was named pSHL. Since the colony's L62 lipase production capacity was 2,500 U / L, it was confirmed that the complete lipase gene was present in the 4.2 kb fragment.
L62 리파제의 DNA 단편을 삽입한 재조합 플라스미드 pSHL의 서열분석을 통하여 2,133 bp의 ORF(open reading frame)를 확인하였다. 이러한 ORF는 711개의 아미노산으로 이루어져 있으며, 분자량이 약 80 kDa으로 추정되는 프리프로리파제(preprolipase)를 암호화하고 있는 것으로 나타났다.An open reading frame (ORF) of 2,133 bp was confirmed by sequencing the recombinant plasmid pSHL into which the DNA fragment of L62 lipase was inserted. This ORF consists of 711 amino acids and has been shown to encode a preprolipase with an estimated molecular weight of about 80 kDa.
L62 리파아제의 프리프로리파제는 실시예 1의 N-말단 아미노산 서열을 근거로 60개의 아미노산으로 이루어진 신호서열(signal sequence), 259개의 아미노산으로 이루어진 프로펩타이드 및 392개의 아미노산으로 이루어진 머추어(mature) 리파제로 구성되어 있음을 추정할 수 있다(도3).Preprolipase of L62 lipase is a lipase consisting of a signal sequence consisting of 60 amino acids, a propeptide consisting of 259 amino acids and a 392 amino acid based on the N-terminal amino acid sequence of Example 1 It can be estimated that it is composed of (Fig. 3).
스타필로코커스 해모리티커스(S. haemolyticus) L62으로부터 생산되는 리파제는 스타필로코커스 속의 다른 균주들이 생산하는 다른 리파제와 어느 정도 유사하였으나, 단백질 수준에서 다음 표 2와 같이 스타필로코커스 아우레우스(S. aureus), 스타필로코커스 에피덜미디스(S. epidermidis) 및 스타필로코커스 하이쿠스(S. hyicus)가 생산하는 리파제와 각각 약 67%, 55% 및 51%의 유사성을 가지므로 신규 효소로 판정되었다.Staphylococcus haemolyticusS. haemolyticusThe lipase produced from L62 was somewhat similar to other lipases produced by other strains of Staphylococcus, but at the protein level, Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis) And Staphylococcus hydrusS. hyicus) Has been identified as a novel enzyme because it has about 67%, 55% and 51% similarity with the lipase produced.
실시예 3: L62 리파제의 대량생산을 위한 재조합 플라스미드의 제조Example 3 Preparation of Recombinant Plasmids for Mass Production of L62 Lipase
PCR(polymerase chain reaction)을 이용하여 리파제 유전자 중 신호서열(signal sequence) 및 프로펩타이드(propeptide)를 제외한 머추어(mature) 리파제 DNA의 증폭을 위해 서열 3에 기재된 머추어(mature) 리파제의 DNA 염기 서열로부터 서열 1 및 서열 2에 기재된 프라이머 P1 및 P2를 제작한 후, PCR을 수행하여 1.15 kb의 머추어(mature) 리파제 유전자를 증폭하였다.DNA nucleotide of the mature lipase as described in SEQ ID NO: 3 for amplification of the mature lipase DNA excluding signal sequence and propeptide of lipase gene using polymerase chain reaction (PCR) After preparing the primers P1 and P2 described in SEQ ID NO: 1 and SEQ ID NO: 2 from the sequence, PCR was performed to amplify the 1.15 kb of mature lipase gene.
그런 다음, 도 4에 나타낸 바와 같이 T7 프로모터를 함유하는 pET22-b(+) 발현 벡터에 증폭된 리파제 유전자를 삽입하고, 이러한 재조합 플라스미드를 pSHML로 명명하였다.Then, the amplified lipase gene was inserted into the pET22-b (+) expression vector containing the T7 promoter as shown in FIG. 4, and this recombinant plasmid was named pSHML.
실시예 4: L62 리파제가 대량생산되는 형질전환된 대장균의 제조Example 4 Preparation of Transformed Escherichia Coli with Mass Production of L62 Lipase
T7 RNA 중합효소를 갖고 있는 대장균 BL21(DE3)을 숙주세포로 하여 실시예 3에서 제조한 플라스미드 pSHML을 도입시켰다. 형질 전환된 대장균을 100 ㎍/㎖의 암피실린(ampicillin)이 함유된 LB배지를 사용하여 37℃에서 배양하였다. 그런 다음, OD 600 nm에서 흡광도가 0.6에 도달하였을 때, IPTG를 최종 농도 1 mM 되도록 첨가하여 리파제 발현을 유도하고, 1시간 간격으로 샘플을 취하여 원심 분리하였다.The plasmid pSHML prepared in Example 3 was introduced using E. coli BL21 (DE3) having a T7 RNA polymerase as a host cell. Transformed E. coli was cultured at 37 ° C. using LB medium containing 100 μg / ml of ampicillin. Then, when the absorbance reached 0.6 at OD 600 nm, IPTG was added to a final concentration of 1 mM to induce lipase expression, and samples were taken at 1 hour intervals and centrifuged.
원심분리하여 얻은 균체를 초음파로 분쇄하여 원심 분리한 후, 용해성 단백질에 대해 12% SDS-PAGE을 행한 결과(도 5a), 전체 단백질 중 리파제의 함량이 약 30%까지 생산되고, IPTG 첨가한지 4시간 이후의 L62 리파제 활성이 80,000 U/ℓ로 최대 활성을 보였다(도 5b).After centrifugation of the cells obtained by centrifugation and centrifugation by ultrasonic, 12% SDS-PAGE of the soluble protein (Fig. 5a), the lipase content of the total protein is produced up to about 30%, IPTG added 4 L62 lipase activity after time showed maximum activity at 80,000 U / L (FIG. 5B).
상기 결과는 대장균 XL1 Blue/pSHL의 L62 리파제 생산량에 비하여 32배정도 증가된 생산 양이며, 스타필로코커스 해모리티커스(S. haemolyticus) L62인 원균에 대해서는 8.2배 증가한 양으로 산업적으로 효소의 이용이 용이할 것으로 사료된다.The result is an increase of about 32 times that of L62 lipase produced by Escherichia coli XL1 Blue / pSHL, and an increase of 8.2 times for S. haemolyticus L62, which is an industrial factor. It is thought to be easy.
따라서, 재조합 플라스미드 pSHML로 형질전환된 대장균 BL21(DE3)를 대장균 BL21(DE3)/pSHML로 명명하고, 이를 생명공학연구소 유전자은행에 1999년 8월 18일자로 기탁하여 수탁번호 KCTC 8956P를 부여받았다.Therefore, Escherichia coli BL21 (DE3) transformed with the recombinant plasmid pSHML was named Escherichia coli BL21 (DE3) / pSHML, and was deposited on August 18, 1999 by the Biotechnology Research Institute Gene Bank to receive accession number KCTC 8956P.
실시예 5: 형질전환된 대장균 BL21(DE3)/pSHML으로부터 생산되는 L62 리파제의 분리Example 5: Isolation of L62 Lipase Produced from Transfected Escherichia Coli BL21 (DE3) / pSHML
상기 실시예 4에서 제조한 형질전환된 대장균 BL21(DE3)/pSHML으로부터 생산되는 L62 리파제는 높은 등전점(pI=9.7)을 갖기 때문에 다음과 같이 간단한 방법으로 순수 분리하였다. 1 mM IPTG 처리 후, 4시간 동안 배양하여 얻은 균체를 초음파 분쇄법으로 파쇄한 후에 원심분리를 통해 상층액을 얻고, 이를 20 mM 트리스/HCl 완충액(pH 8.8)으로 투석하였다.Since L62 lipase produced from the transformed Escherichia coli BL21 (DE3) / pSHML prepared in Example 4 has a high isoelectric point (pI = 9.7), it was purely separated as follows. After 1 mM IPTG treatment, cells obtained by incubation for 4 hours were crushed by ultrasonic grinding, and then the supernatant was obtained by centrifugation, and dialyzed with 20 mM Tris / HCl buffer (pH 8.8).
음이온성 교환수지인 리소스(Resourse) Q 컬럼(pH 8.8)에서는 대부분의 대장균 단백질은 결합하지만, L62 리파제는 결합하지 않으므로, 리소스(Resourse) Q 컬럼을 한번 통과시킴으로써 간단히 리파제를 순수 분리하였고, 그 결과를 도 6에 나타내었다.Resourse Q column (pH 8.8), an anionic exchange resin, binds most E. coli proteins but does not bind L62 lipase, so the lipase was purely separated by one pass through a Resourse Q column. Is shown in FIG. 6.
실시예 6: L62 리파제의 트리올레인(triolein)에 대한 위치 특이성Example 6: Location Specificity for Triolein of L62 Lipase
상기 실시예 5에서 분리된 L62 리파제의 트리올레인에 대한 위치 특이성을 확인한 결과, L62 리파제는 트리올레인의 1,3-위치를 특이적으로 분해하는 1,3-위치 특이적 리파제임을 확인하였고 그 결과를 도 7에 나타내었다. 즉, L62 리파제를 처리하면 트리올레인으로부터 1(3)-모노올레인 및 1,2(2,3)-다이올레인을 얻을 수 있다.As a result of confirming the position specificity of triolein of L62 lipase isolated in Example 5, it was confirmed that L62 lipase is a 1,3-position specific lipase that specifically degrades the 1,3-position of triolein. The results are shown in FIG. In other words, treatment with L62 lipase yields 1 (3) -monoolein and 1,2 (2,3) -diolane from triolein.
실시예 7: L62 리파제 활성에 대한 계면활성제의 영향Example 7: Effect of Surfactant on L62 Lipase Activity
L62 리파제 활성에 대한 계면활성제의 영향을 검토하기 위하여 각종 계면활성제를 1 부피% 및 5 부피%가 되도록 L62 리파제액에 첨가하여 상온에서 10분간 방치한 후, L62 리파제의 활성을 pH 스태트(stat)법으로 측정하였다. 그 결과를 다음 표 3에 나타내고, 비이온성 계면활성제에 의해 L62 리파제의 활성이 4∼6배정도 증가했으며, SDS와 같은 음이온성 계면활성제는 효소의 활성을 감소시켰다. 또한, 담즙산(bile salt)인 타우로콜레이트(taurocholate)도 리파제의 활성을 증가시켰다.In order to examine the effect of surfactant on L62 lipase activity, various surfactants were added to L62 lipase solution to 1% by volume and 5% by volume, and allowed to stand at room temperature for 10 minutes, and then the activity of L62 lipase was changed to pH stat. It was measured by the method. The results are shown in Table 3 below, and the activity of L62 lipase was increased by 4 to 6 times by nonionic surfactant, and anionic surfactants such as SDS decreased the activity of the enzyme. In addition, taurocholate, a bile acid, also increased lipase activity.
한편, 계면활성제가 처리된 L62 리파제의 기질 특이성을 조사한 결과, 대부분의 트리글리세라이드에 대한 효소 활성이 1.5∼3배 증가하였으며, 특히, 불포화 지방산을 함유한 트리리놀레인(trilinolein, C18:2) 및 트리리놀레인(trilinolenin, C18:3)에 대한 효소의 활성은 각각 10배 및 3배 증가하였다. 또한, 트윈 80이 처리된 L62 리파제는 트리글리세라이드 중에서 트리프로피오닌(C3), 트리뷰트린(C4), 트리미리스틴(C14) 및 트리리롤레인(C18:2)에 대해 무처리된 L62 리파제보다 효소 활성이 커졌으며, 그 결과를 표 4에 나타내었다.On the other hand, as a result of investigating the substrate specificity of the surfactant-treated L62 lipase, the enzyme activity for most triglycerides increased 1.5 to 3 times, especially trilinolein (C18: 2) containing unsaturated fatty acids and Enzyme activity on trilinolenin (C18: 3) increased 10-fold and 3-fold, respectively. In addition, L62 lipase treated with Tween 80 is more effective than L62 lipase untreated for tripropionine (C3), tributrin (C4), trimyristin (C14) and tririlollein (C18: 2) in triglycerides. Enzyme activity was increased, the results are shown in Table 4.
또한, 트윈 80이 처리된 L62 리파제는 천연유지 중 리놀레산(linoleic acid, C18:3)이 다량 함유된 대두유(리놀레산 53% 함유), 목화유(리놀레산 47% 함유), 맥아유(wheat germ oil, 리놀레산 42% 함유) 및 옥수수유(리놀레산 39% 함유)에 대한 효소의 활성이 무처리된 L62 리파제에 비해 5∼7배 증가하였다(도 8). 상기와 같이 계면활성제가 처리된 L62 리파제의 경우, 효소의 기질특이성이 변하는 것을 산업적 목적에 맞게 이용할 수 있다는 장점이 있다.In addition, L62 lipase treated with Tween 80 contains soybean oil (containing 53% linoleic acid) containing 53% linoleic acid (C18: 3), cotton oil (containing 47% linoleic acid), malt oil (wheat germ oil) Enzyme activity against linoleic acid 42%) and corn oil (containing 39% linoleic acid) was increased 5-7 fold compared to untreated L62 lipase (FIG. 8). In the case of the L62 lipase treated with the surfactant as described above, there is an advantage that the substrate specificity of the enzyme can be used for industrial purposes.
상술한 바와 같이, 본 발명은 계면활성제에 활성화되는 저온성 L62 리파제를 생산하는 신균주 스타필로코커스 헤모리티커스(S. haemolyticus) L62 및 형질전환된 대장균 BL21(DE3)/pSHML을 이용한 L1 리파제의 대량생산법에 관한 것으로, L62리파제는 비이온성 계면활성제에 의해 효소의 활성이 5∼6배 증가하고, 저온에서 효소의 활성이 우수한 것으로 밝혀져서, 고온에서 불안정한 화합물 및 불포화 지방산이 함유된 천연유지를 가수분해하여 고가의 불포화 지방산을 생산하거나, 에스테르 및 펩타이드를 합성하는 공정 등에 유용하게 사용될 수 있다.As described above, the present invention provides a novel strain L. lipase using S. haemolyticus L62 and transformed Escherichia coli BL21 (DE3) / pSHML to produce a low temperature L62 lipase that is activated to a surfactant. L62 lipase was found to have a 5 to 6-fold increase in the activity of the enzyme by a nonionic surfactant, and showed excellent activity of the enzyme at low temperatures, and thus contained unstable compounds and unsaturated fatty acids at high temperatures. It can be usefully used for hydrolyzing oils and fats to produce expensive unsaturated fatty acids, or for synthesis of esters and peptides.
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