KR102637345B1 - Novel phospholipase A2 derived from Bacillus megaterium with enhanced phospholipid hydrolyzation activity - Google Patents
Novel phospholipase A2 derived from Bacillus megaterium with enhanced phospholipid hydrolyzation activity Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01004—Phospholipase A2 (3.1.1.4)
Abstract
본 발명은 인지질 분해능이 개선된 Bacillus megaterium 유래 신규 포스포리파아제 A2(phopholipase A2) 효소, 이를 암호화하는 폴리뉴클레오타이드 등에 관한 것으로서, 실수유발 중합효소 연쇄 반응(Error-prone PCR)을 통해 형질전환함으로써 상기 포스포리파아제 A2를 수득할 수 있으며, 상기 포스포리파아제 A2는 레시틴의 가수분해 활성이 우수하여 이를 사용한 산업에서 유용하게 활용될 수 있고 박테리아 발현 벡터를 이용하여 대량생산이 가능하기 때문에 생산 비용 측면에서 경쟁력을 가질 수 있다.The present invention relates to a novel phospholipase A2 enzyme derived from Bacillus megaterium with improved phospholipid decomposition ability, a polynucleotide encoding the same, and the phospholipase A2 enzyme is obtained by transformation through error-prone PCR. Phospholipase A2 can be obtained, and phospholipase A2 has excellent lecithin hydrolytic activity, so it can be usefully utilized in industries using it, and mass production is possible using bacterial expression vectors, making it competitive in terms of production cost. You can have
Description
본 발명은 인지질 분해능이 개선된 Bacillus megaterium 유래 신규 포스포리파아제 A2(phospholipase A2) 등에 관한 것이다. The present invention relates to a novel phospholipase A2 derived from Bacillus megaterium with improved phospholipid decomposition ability, etc.
효소는 현재 다양한 산업에서 많이 사용되고 있다. 효소는 화학적 방법을 통한 생산 방식보다 저렴하고 특이적 반응을 통해 생산물들의 품질이 좋다는 장점이 있다. 또한 단순 발효를 통한 생산보다 효소 정제를 통한 생산의 경우 특정 물질을 생산하기에 용이하다. 연평균 세계 효소 시장 규모는 지속적인 성장을 이루고 있으며 한국이 포함된 아시아 시장의 성장률이 가장 크다. Enzymes are currently widely used in various industries. Enzymes have the advantage of being cheaper than chemical production methods and producing better quality products through specific reactions. In addition, production through enzyme purification is easier to produce specific substances than production through simple fermentation. The average annual global enzyme market size continues to grow, with the largest growth rate in the Asian market, including Korea.
현재 실생활과 필수인 식품, 환경, 제지, 정유, 세제, 축산 등에서 효소에 대한 큰 수요를 보이고 있다. 초기에는 화학적인 방법으로 필요 물질을 합성하여 준비과정, 기기설비, 정제등에서 높은 비용이 요구되었다. 이후 단순 화학적인 방법을 통한 물질합성이 아닌 효소사용 생산공정을 통해 필요 비용이 크게 줄어들었으며, 각 기업들은 생산비용을 절감하여 큰 경쟁력을 갖게되었다. 각 산업분야에서 효소이용 물질 생산과정시 요구되는 효소의 반응 조건은 차이가 클 수 있다. 온도, pH등과 같은 반응 조건이나 기질들의 특성이 모두 달라 효소가 최대 효율을 갖는 조건에서 물질을 생산하는 것이 큰 관건이다. 이렇기 때문에 각 산업분야에서 요구되는 생산조건에서 최대의 효율을 갖는 효소의 수요가 증가하고 있다.Currently, there is a great demand for enzymes in daily life and essential areas such as food, environment, papermaking, oil refining, detergents, and livestock farming. In the beginning, high costs were required for the preparation process, equipment, and purification by synthesizing the necessary substances through chemical methods. Since then, the necessary costs have been greatly reduced through a production process using enzymes rather than material synthesis through simple chemical methods, and each company has gained great competitiveness by reducing production costs. The enzyme reaction conditions required during the production process of enzyme-using substances in each industrial field can vary significantly. Reaction conditions such as temperature, pH, etc. and characteristics of substrates are all different, so producing substances under conditions where enzymes have maximum efficiency is a major key. For this reason, the demand for enzymes with maximum efficiency under the production conditions required in each industrial field is increasing.
포스포리파아제 A2의 경우 다른 포스포리파아제보다 굉장히 넓은 분야에서 사용되고 있다. 식품 산업에서는 치즈, 아이스크림과 같은 유제품의 공정, 계란 노른자를 유화시키는 마요네즈 공정과정에서 사용되고, 기름 정제 공정의 탈검과정, 미생물에 대해 항균활성 증대를 위해서도 사용이 되고 있다.Phospholipase A2 is used in a much wider field than other phospholipases. In the food industry, it is used in the processing of dairy products such as cheese and ice cream, in the mayonnaise process to emulsify egg yolks, and in the degumming process of oil refining processes and to increase antibacterial activity against microorganisms.
현재 기존 산업에서 이용되는 효소 시장은 굉장히 크지만, 보다 개선된 효소의 제시를 통해 해당 산업에 종사하는 기업들의 부흥과 동일 산업내의 기업들보다 높은 경쟁력을 확보할 수 있다. 또한 포스포리파아제 A2의 대량 생산과 정제를 통해 효소의 가격 인하와 동시에 생산가 인하를 기대할 수 있다. 때문에 여러 조건에서 최적화되고 안정적인 효소의 확보에 대한 수요를 충족시킬 수 있는 신규 포스포리파아제 A2의 개발이 시급한 실정이다.Currently, the market for enzymes used in existing industries is very large, but the presentation of improved enzymes can help companies in the industry revive and secure higher competitiveness than companies in the same industry. In addition, through mass production and purification of phospholipase A2, it is expected that the price of the enzyme will be reduced and the production price will be reduced at the same time. Therefore, there is an urgent need to develop a new phospholipase A2 that can meet the demand for an optimized and stable enzyme under various conditions.
이에, 본 발명자들은 실수유발 중합효소 연쇄 반응(error-prone PCR)을 통해 인지질 분해능이 개선된 바실러스 메가테리움(Bacillus megaterium) 유래 신규 포스포리파아제 A2를 발굴하여, 본 발명을 완성하였다.Accordingly, the present inventors discovered a new phospholipase A2 derived from Bacillus megaterium with improved phospholipid decomposition ability through error-prone polymerase chain reaction (error-prone PCR) and completed the present invention.
본 발명이 이루고자 하는 기술적 과제는 바실러스 메가테리움(Bacillus megaterium) 유래의 서열번호 3 또는 4의 아미노산 서열로 이루어진, 인지질 분해활성을 가지는 포스포리파아제 A2(phospholipase A2) 효소, 이를 암호화하는 뉴클레오티드, 이를 포함하는 발현 벡터, 이를 포함하는 형질전환체를 제공하는 것이다.The technical problem to be achieved by the present invention is to provide a phospholipase A2 enzyme with phospholipid-degrading activity, consisting of the amino acid sequence of SEQ ID NO: 3 or 4 derived from Bacillus megaterium , a nucleotide encoding the same, and An expression vector containing the same and a transformant containing the same are provided.
또한, 본 발명의 다른 목적은 바실러스 메가테리움(Bacillus megaterium) 유래의 서열번호 3 또는 4의 아미노산 서열로 이루어진 포스포리파아제 A2(phospholipase A2); 또는 이를 암호화하는 서열번호 1 또는 2의 염기서열로 이루어진 유전자를 유효성분으로 포함하는, 인지질 분해용 조성물을 제공하는 것이다.In addition, another object of the present invention is phospholipase A2 consisting of the amino acid sequence of SEQ ID NO: 3 or 4 derived from Bacillus megaterium ; Alternatively, a composition for decomposing phospholipids is provided, which contains as an active ingredient a gene consisting of the base sequence of SEQ ID NO: 1 or 2 encoding the same.
또한, 본 발명의 또 다른 목적은 바실러스 메가테리움(Bacillus megaterium) 유래의 서열번호 3 또는 4의 아미노산 서열로 이루어진, 인지질 분해활성을 가지는 포스포리파아제 A2에 레시틴을 처리하는 단계를 포함하는, 리소레시틴의 생산 방법을 제공하는 것이다.In addition, another object of the present invention is to process phospholipase A2, which has phospholipid decomposing activity and consists of the amino acid sequence of SEQ ID NO: 3 or 4 from Bacillus megaterium , with lecithin. To provide a method for producing lecithin.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당해 기술분야의 통상의 기술자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.
상기 과제를 해결하기 위하여, 본 발명은 바실러스 메가테리움(Bacillus megaterium) 유래의 서열번호 3 또는 4의 아미노산 서열로 이루어진, 인지질 분해활성을 가지는 포스포리파아제 A2(phospholipase A2) 효소를 제공한다.In order to solve the above problems, the present invention provides a phospholipase A2 enzyme having phospholipid decomposing activity, consisting of the amino acid sequence of SEQ ID NO: 3 or 4 derived from Bacillus megaterium .
본 발명의 일 구현예로서, 상기 서열번호 4의 아미노산 서열로 이루어진 포스포리파아제 A2는 서열번호 3의 10번째 위치에 상응하는 아미노산인 트레오닌이 알라닌으로 치환되고, 47번째 위치에 상응하는 아미노산인 아스파르트산이 글리신으로 치환된 것일 수 있으며, 상기 서열번호 4의 아미노산 서열로 이루어진 포스포리파아제 A2는 상기 서열번호 3의 아미노산 서열로 이루어진 포스포리파아제 A2보다 인지질 분해능이 더 우수할 수 있다.In one embodiment of the present invention, phospholipase A2 consisting of the amino acid sequence of SEQ ID NO: 4 is prepared by replacing threonine, the amino acid corresponding to the 10th position of SEQ ID NO: 3, with alanine, and aspart, the amino acid corresponding to the 47th position. The acid may be substituted with glycine, and phospholipase A2 composed of the amino acid sequence of SEQ ID NO: 4 may have a better phospholipid decomposition ability than phospholipase A2 composed of the amino acid sequence of SEQ ID NO: 3.
본 발명의 다른 구현예로서, 상기 조성물은 레시틴(lecithin)을 가수분해하여 리소레시틴(lysolecithin)을 생산하는 것일 수 있다.As another embodiment of the present invention, the composition may produce lysolecithin by hydrolyzing lecithin.
본 발명의 또 다른 구현예로서, 상기 포스포리파아제 A2 효소는 32.5℃pH 8.0에서 최적의 인지질 분해활성을 가지는 것일 수 있다.As another embodiment of the present invention, the phospholipase A2 enzyme may have optimal phospholipid decomposition activity at 32.5°C and pH 8.0.
또한, 본 발명은 상기 포스포리파아제 A2를 암호화하는, 서열번호 1 또는 2의 염기서열로 이루어진, 폴리뉴클레오티드를 제공한다.In addition, the present invention provides a polynucleotide consisting of the base sequence of SEQ ID NO: 1 or 2, encoding the phospholipase A2.
또한, 본 발명은 상기 폴리뉴클레오티드를 포함하는 발현 벡터를 제공한다.Additionally, the present invention provides an expression vector containing the above polynucleotide.
또한, 본 발명은 상기 발현 벡터를 포함하는 형질전환체를 제공한다.Additionally, the present invention provides a transformant containing the above expression vector.
또한, 본 발명은 바실러스 메가테리움(Bacillus megaterium) 유래의 서열번호 3 또는 4의 아미노산 서열로 이루어진 포스포리파아제 A2(phospholipase A2); 또는 이를 암호화하는 서열번호 1 또는 2의 염기서열로 이루어진 유전자를 유효성분으로 포함하는, 인지질 분해용 조성물을 제공한다.In addition, the present invention provides phospholipase A2 consisting of the amino acid sequence of SEQ ID NO: 3 or 4 derived from Bacillus megaterium ; Alternatively, a composition for decomposing phospholipids is provided, which contains as an active ingredient a gene consisting of the base sequence of SEQ ID NO: 1 or 2 encoding the same.
또한, 본 발명은 바실러스 메가테리움(Bacillus megaterium) 유래의 서열번호 3 또는 4의 아미노산 서열로 이루어진, 인지질 분해활성을 가지는 포스포리파아제 A2에 기질을 처리하는 단계를 포함하는, 리소레시틴의 생산 방법을 제공한다.In addition, the present invention provides a method for producing lysolecithin, comprising the step of treating the substrate with phospholipase A2, which has phospholipid-degrading activity and consists of the amino acid sequence of SEQ ID NO: 3 or 4 derived from Bacillus megaterium. provides.
본 발명의 일 구현예로서, 상기 포스포리파아제 A2는 서열번호 1 또는 2의 염기서열로 이루어진 유전자를 발현하는 재조합 대장균을 배양하여 수득한 것일 수 있다.As an embodiment of the present invention, the phospholipase A2 may be obtained by culturing recombinant E. coli expressing a gene consisting of the base sequence of SEQ ID NO: 1 or 2.
본 발명의 다른 구현예로서, 상기 단계는 20 내지 37.5℃에서 수행하는 것일 수 있으며, 바람직하게는 32.5℃에서 수행되는 것일 수 있다.As another embodiment of the present invention, the step may be performed at 20 to 37.5°C, and preferably at 32.5°C.
본 발명의 또 다른 구현예로서, 25 내지 35℃에서 기질 없이 상기 포스포리파아제 A2를 첨가하고 30분 내지 2시간 후에, 상기 단계를 수행하는 것일 수 있다.As another embodiment of the present invention, the above step may be performed 30 minutes to 2 hours after adding the phospholipase A2 without a substrate at 25 to 35°C.
본 발명의 또 다른 구현예로서, 상기 단계는 pH 7.5 내지 8.5에서 수행하는 것일 수 있으며, 바람직하게는 pH 8.0에서 수행되는 것일 수 있다.As another embodiment of the present invention, the step may be performed at pH 7.5 to 8.5, preferably at pH 8.0.
본 발명의 또 다른 구현예로서, pH 7.5 내지 8.5에서 기질 없이 상기 포스포리파아제 A2를 첨가하고 30분 내지 2시간 후에, 상기 단계를 수행하는 것일 수 있다.As another embodiment of the present invention, the step may be performed 30 minutes to 2 hours after adding the phospholipase A2 without a substrate at pH 7.5 to 8.5.
본 발명의 또 다른 구현예로서, 상기 기질의 최소 농도는 실험 100 μL버퍼당 0.1 μg, 즉, 1 μg/mL인 것일 수 있다.As another embodiment of the present invention, the minimum concentration of the substrate may be 0.1 μg per 100 μL experimental buffer, that is, 1 μg/mL.
본 발명은 인지질 분해능이 개선된 Bacillus megaterium 유래 신규 포스포리파아제 A2(phopholipase A2) 효소, 이를 암호화하는 폴리뉴클레오타이드 등에 관한 것으로서, 실수유발 중합효소 연쇄 반응(Error-prone PCR)을 통해 형질전환함으로써 상기 포스포리파아제 A2를 수득할 수 있으며, 상기 포스포리파아제 A2는 레시틴의 가수분해 활성이 우수하여 이를 사용한 산업에서 유용하게 활용될 수 있고 박테리아 발현 벡터를 이용하여 대량생산이 가능하기 때문에 생산 비용 측면에서 경쟁력을 가질 수 있다.The present invention relates to a novel phospholipase A2 enzyme derived from Bacillus megaterium with improved phospholipid decomposition ability, a polynucleotide encoding the same, and the phospholipase A2 enzyme is obtained by transformation through error-prone PCR. Phospholipase A2 can be obtained, and phospholipase A2 has excellent lecithin hydrolytic activity, so it can be usefully utilized in industries using it, and mass production is possible using bacterial expression vectors, making it competitive in terms of production cost. You can have
도 1은 본 발명의 실시예 3에서 형질전환된 각 효소들의 EEPC 분해 활성을 비교한 결과이다.
도 2은 본 발명의 실시예 4에서 수행한 발현 조건에서 B. megaterium 유래 포스포리파아제 A2의 발현량을 확인한 결과이다.
도 3은 본 발명의 실시예 5에서 수득한 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2, 실시예 2에서 수득한 B. megaterium 유래 포스포리파아제 A2, 상업적으로 사용되고 있는 Novozymes사의 LowP의 효소 활성을 비교한 결과이다.
도 4는 본 발명의 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2의 기질량에 따른 활성을 확인한 결과이다.
도 5는 본 발명의 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2의 온도에 따른 활성을 확인한 결과이다.
도 6은 본 발명의 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2을 각각 다른 온도에서 전보온 후 잔효성을 확인한 결과이다.
도 7은 본 발명의 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2의 pH에 따른 활성을 확인한 결과이다.
도 8은 본 발명의 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2을 각각 다른 pH에서 전보온 후 잔효성을 확인한 결과이다.Figure 1 shows the results of comparing the EEPC decomposition activities of each enzyme transformed in Example 3 of the present invention.
Figure 2 shows the results of confirming the expression level of B. megaterium- derived phospholipase A2 under the expression conditions performed in Example 4 of the present invention.
Figure 3 shows B. megaterium- derived phospholipase A2 with improved phospholipid degrading ability obtained in Example 5 of the present invention, B. megaterium- derived phospholipase A2 obtained in Example 2, and the commercially used enzyme LowP from Novozymes. This is the result of comparing activity.
Figure 4 shows the results of confirming the activity of B. megaterium- derived phospholipase A2 with improved phospholipid decomposition ability of the present invention according to the amount of substrate.
Figure 5 shows the results of confirming the temperature-dependent activity of B. megaterium- derived phospholipase A2 with improved phospholipid decomposition ability of the present invention.
Figure 6 shows the results of confirming the residual effectiveness of B. megaterium- derived phospholipase A2, which has improved phospholipid decomposition ability of the present invention, after preheating at different temperatures.
Figure 7 shows the results of confirming the activity of B. megaterium- derived phospholipase A2 with improved phospholipid decomposition ability of the present invention according to pH.
Figure 8 shows the results of confirming the residual effectiveness of B. megaterium- derived phospholipase A2, which has improved phospholipid decomposition ability of the present invention, after preheating at different pH.
본 발명자들은 레시틴을 기질로 하여 포스포리파아제 A2을 이용하여 지방산과 리소레시틴을 생합성하는 방법에 대해 연구한 결과, 실수유발 중합효소 연쇄 반응(Error-prone PCR)을 통해 형질전환함으로써 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2를 개발하였다.The present inventors studied a method for biosynthesizing fatty acids and lysolecithin using phospholipase A2 using lecithin as a substrate, and found that phospholipid decomposition ability was improved by transformation through error-prone PCR. Phospholipase A2 derived from B. megaterium was developed.
보다 구체적으로, 우수한 포스포리파아제 A2를 가지는 미생물을 선별하기 위해 고농도의 인지질 배지에서 높은 인지질 분해 활성을 보이는 미생물을 확인한 후, 확보한 미생물로부터 후보 포스포리파아제 A2의 유전자를 확보한 다음, 이를 미생물에서 발현시켜 인지질 분해 활성을 비교하였다. 실수유발 중합효소 연쇄 반응 돌연변이화법을 통해 선별된 포스포리파아제 A2의 돌연변이 포스포리파아제 A2 유전자를 확보하였다. 이를 잘 알려진 미생물을 통해 대량 발현조건에서 활성이 개선된 포스포리파아제 A2를 발현한 후 정제하여 활성이 증가한 것을 확인하였다.More specifically, in order to select microorganisms with excellent phospholipase A2, microorganisms showing high phospholipid decomposition activity in a high-concentration phospholipid medium were identified, and then candidate phospholipase A2 genes were obtained from the obtained microorganisms, which were then transferred to the microorganisms. were expressed in and the phospholipid decomposition activity was compared. A mutant phospholipase A2 gene of phospholipase A2 selected was obtained through mistake-prone polymerase chain reaction mutagenesis. Phospholipase A2 with improved activity was expressed under mass expression conditions using a well-known microorganism and then purified to confirm that the activity increased.
또한, 상기 형질전환으로 확보한 활성이 개선된 B. megaterium 유래 포스포리파아제 A2의 특성을 밝히기 위해 기질의 농도, 온도, pH에서의 활성을 확인하였고 온도와 pH에 대해 안정성을 확인하였으며 기존의 B. megaterium 유래 포스포리파아제 A2와 시중에 판매되고 있는 포스포리파아제 A2의 비교를 통해 본 발명의 포스포리파아제 A2의 우수한 활성과 사용 조건을 확인하였다.In addition, in order to reveal the characteristics of B. megaterium- derived phospholipase A2 with improved activity obtained through the above transformation, the activity was confirmed at substrate concentration, temperature, and pH, and stability was confirmed for temperature and pH, and the existing B Through a comparison between phospholipase A2 derived from megaterium and commercially available phospholipase A2, the excellent activity and usage conditions of phospholipase A2 of the present invention were confirmed.
이에, 본 발명은 바실러스 메가테리움(Bacillus megaterium) 유래의 서열번호 3 또는 4의 아미노산 서열로 이루어진 포스포리파아제 A2(phospholipase A2); 또는 이를 암호화하는 서열번호 1 또는 2의 염기서열로 이루어진 폴리뉴클레오티드를 제공한다.Accordingly, the present invention provides phospholipase A2 consisting of the amino acid sequence of SEQ ID NO: 3 or 4 derived from Bacillus megaterium; Alternatively, a polynucleotide consisting of the base sequence of SEQ ID NO: 1 or 2 encoding the same is provided.
본 발명에서, 용어 “포스포리파아제(Phospholipase)”는 포스포리파아제는 인지질을 가수분해하는 효소로서 레시티나아제라고도 한다. 포스포리파아제는 인지질을 분해하는 위치에 따라 A1, A2, B, C, D 로 분류되며, 상기 포스포리파아제 중 본 발명의 “포스포리파아제 A2(PLA2)”는 세포막 구성 지질인 포스포리피드의 글리세린의 BDP 결합하는 지방산을 유리시켜 리조포스파티드로 만들기 때문에 세포조직의 파괴, 용혈작용 및 촉매 작용 등을 일으키는 것으로 알려져 있다.In the present invention, the term “phospholipase” refers to phospholipase, which is an enzyme that hydrolyzes phospholipids and is also called lecithinase. Phospholipases are classified into A1, A2, B, C, and D depending on the location where they decompose phospholipids. Among the phospholipases, “phospholipase A2 (PLA2)” of the present invention is a phospholipid of phospholipids, which are lipids that make up cell membranes. It is known to cause destruction of cell tissue, hemolysis, and catalytic action because it liberates the fatty acid that binds to BDP of glycerin and turns it into lysophosphatide.
본 발명은 상기 염기서열 및 아미노산 서열의 상동체를 포함하며, 구체적으로 상기 폴리뉴클레오티드 및 아미노산은 각각 서열번호 1 또는 2, 서열번호 3 또는 4의 각각의 서열과 각각 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가질 수 있다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.The present invention includes homologs of the base sequence and amino acid sequence, and specifically, the polynucleotide and amino acid are each 70% or more of the respective sequences of SEQ ID NO: 1 or 2, SEQ ID NO: 3 or 4, more preferably It may have sequence homology of at least 80%, more preferably at least 90%, and most preferably at least 95%. The “% sequence homology” for a polynucleotide is determined by comparing a comparison region with two optimally aligned sequences, where a portion of the polynucleotide sequence in the comparison region is a reference sequence (additions or deletions) for the optimal alignment of the two sequences. may contain additions or deletions (i.e. gaps) compared to those that do not contain .
또한, 본 발명은 바실러스 메가테리움(B. megaterium) 유래의 서열번호 3 또는 4의 아미노산 서열로 이루어진 포스포리파아제 A2(phospholipase A2)를 암호화하는 서열번호 1 또는 2의 염기서열로 이루어진 유전자를 포함하는 재조합 벡터, 또는 상기 재조합 벡터로 형질전환된 형질전환체 등을 제공한다.In addition, the present invention includes a gene consisting of the base sequence of SEQ ID NO: 1 or 2, which encodes phospholipase A2, which is derived from B. megaterium and consists of the amino acid sequence of SEQ ID NO: 3 or 4. A recombinant vector or a transformant transformed with the recombinant vector is provided.
본 발명에서, 용어 "재조합"은 세포가 이종의 핵산을 복제하거나, 상기 핵산을 발현하거나 또는 펩티드, 이종의 펩티드 또는 이종의 핵산에 의해 암호된 단백질을 발현하는 세포를 지칭하는 것이다. 재조합 세포는 상기 세포의 천연 형태에서는 발견되지 않는 유전자 또는 유전자 절편을, 센스 또는 안티센스 형태 중 하나로 발현할 수 있다. 또한 재조합 세포는 천연 상태의 세포에서 발견되는 유전자를 발현할 수 있으며, 그러나 상기 유전자는 변형된 것으로써 인위적인 수단에 의해 세포 내 재도입된 것이다.As used herein, the term “recombinant” refers to a cell that replicates a heterologous nucleic acid, expresses a heterologous nucleic acid, or expresses a peptide, a heterologous peptide, or a protein encoded by a heterologous nucleic acid. Recombinant cells can express genes or gene segments that are not found in the natural form of the cell, either in sense or antisense form. Additionally, recombinant cells can express genes found in cells in their natural state, but the genes have been modified and reintroduced into the cells by artificial means.
본 발명에서, 용어 "벡터"는 세포 내로 전달하는 DNA 단편(들), 핵산 분자를 지칭할 때 사용된다. 벡터는 DNA를 복제시키고, 숙주세포에서 독립적으로 재생산될 수 있다. 용어 "전달체"는 흔히 "벡터"와 호환하여 사용된다. 용어 "발현 벡터"는 목적한 코딩 서열과, 특정 숙주 생물에서 작동가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다.In the present invention, the term “vector” is used to refer to a DNA fragment(s) or nucleic acid molecule that is delivered into a cell. Vectors replicate DNA and can reproduce independently in host cells. The term “vector” is often used interchangeably with “vector”. The term “expression vector” refers to a recombinant DNA molecule containing a coding sequence of interest and an appropriate nucleic acid sequence necessary to express the operably linked coding sequence in a particular host organism.
본 발명에서 상기 재조합 벡터는 상기 포스포리파아제 A2를 암호화하는 유전자의 효율적인 발현을 위해 발현 벡터를 사용하는 것이 바람직하다. 본 발명에서 "발현 벡터"란 적당한 숙주세포에서 목적 펩타이드를 발현할 수 있는 재조합 벡터로서, 유전자 삽입물이 발현되도록 작동하게 연결된 필수적인 조절 요소를 포함하는 유전자 제작물을 말한다. 본 발명의 발현 벡터는 적합한 발현 벡터가 일반적으로 가지고 있는 요소로서 프로모터, 오퍼레이터, 개시 코돈 같은 발현조절 요소들을 포함한다. 개시 코돈 및 종결 코돈은 일반적으로 폴리펩타이드를 암호화하는 뉴클레오티드 서열의 일부로 간주되며, 유전자 제작물이 투여되었을 때 개체에서 반드시 작용을 나타내야 하며 코딩 서열과 인프레임(in frame)에 있어야 한다. 벡터의 프로모터는 구성적 또는 유도성 일 수 있다.In the present invention, it is preferable to use an expression vector as the recombinant vector for efficient expression of the gene encoding the phospholipase A2. In the present invention, “expression vector” refers to a recombinant vector capable of expressing a target peptide in a suitable host cell, and refers to a gene construct containing essential regulatory elements operatively linked to express the gene insert. The expression vector of the present invention includes expression control elements such as a promoter, operator, and start codon, which are elements that suitable expression vectors generally have. Initiation codons and stop codons are generally considered to be part of the nucleotide sequence that encodes a polypeptide and must be functional in the subject when the genetic construct is administered and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible.
또한, 세포 배양액으로부터 단백질의 분리를 촉진하기 위하여 융합 폴리펩타이드의 배출을 위한 시그널 서열을 포함할 수 있다. 특이적인 개시 시그널은 또한 삽입된 핵산 서열의 효율적인 번역에 필요할 수도 있다. 이들 시그널은 ATG 개시코돈 및 인접한 서열들을 포함한다. 어떤 경우에는, ATG 개시 코돈을 포함할 수 있는 외인성 번역 조절 시그널이 제공되어야 한다. 이들 외인성 번역 조절 시그널들 및 개시 코돈들은 다양한 천연 및 합성 공급원일 수 있다. 발현 효율은 적당한 전사 또는 번역 강화 인자의 도입에 의하여 증가될 수 있다.Additionally, it may include a signal sequence for release of the fusion polypeptide to promote separation of the protein from the cell culture medium. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG start codon and adjacent sequences. In some cases, an exogenous translation control signal, which may include an ATG initiation codon, must be provided. These exogenous translation control signals and initiation codons can be from a variety of natural and synthetic sources. Expression efficiency can be increased by introduction of appropriate transcription or translation enhancers.
발현 벡터는 통상의 모든 발현 벡터를 사용할 수 있다. 예를 들어, 플라스미드 DNA, 파아지 DNA 등이 사용될 수 있다. 플라스미드 DNA의 구체적인 예로는 pUC18, pIDTSAMRT-AMP 같은 상업적인 플라스미드를 포함한다. 본 발명에 사용될 수 있는 플라스미드의 다른 예로는 대장균 유래 플라스미드(pYG601BR322, pBR325, pUC118 및 pUC119), 바실러스 서브틸리스(Bacillus subtilis)-유래 플라스미드(pUB110 및 pTP5) 및 효모-유래 플라스미드(YEp13, YEp24 및 YCp50)가 있다. 파아지 DNA의 구체적인 예로는 λ파아지(Charon4A, Charon21A, EMBL3, EMBL4, λλ및 λ가 있다. 또한, 리트로바이러스(retrovirus), 아데노바이러스(adenovirus) 또는 백시니아 바이러스(vaccinia virus)와 같은 동물 바이러스, 배큘로바이러스(baculovirus)와 같은 곤충 바이러스가 또한 사용될 수 있다. 이러한 발현 벡터는 숙주 세포에 따라서 단백질의 발현량과 수식 등이 다르게 나타나므로, 목적에 가장 적합한 숙주세포를 선택하여 사용하면 되며 상기 예시에 제한되지 않는다.Any common expression vector can be used as the expression vector. For example, plasmid DNA, phage DNA, etc. can be used. Specific examples of plasmid DNA include commercial plasmids such as pUC18 and pIDTSAMRT-AMP. Other examples of plasmids that can be used in the present invention include E. coli-derived plasmids (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis -derived plasmids (pUB110 and pTP5), and yeast-derived plasmids (YEp13, YEp24 and YCp50). Specific examples of phage DNA include λ phages (Charon4A, Charon21A, EMBL3, EMBL4, λλ, and λ). Additionally, animal viruses such as retrovirus, adenovirus, or vaccinia virus, and bacules Insect viruses such as baculovirus can also be used. Since these expression vectors have different protein expression levels and modifications depending on the host cell, the host cell most suitable for the purpose can be selected and used, limited to the examples above. It doesn't work.
한편, 본 발명의 형질전환체는 본 발명의 재조합 벡터를 적합한 숙주 속에 도입함으로써 얻게 된다. 숙주의 종류로는 에셰리키아(Escherichia)속, 슈도모나스(Pseudomonas)속, 랄스토니아(Ralstonia)속, 알칼리게네스(Alcaligenes)속, 코마모나스(Comamonas)속, 버크홀데리아(Burkholderia)속, 아그로박테리움(Agrobacterium)속, 플라보박테리움(Flabobacterium)속, 비브리오(Vibrio)속, 엔테로박터(Enterobacter)속, 리조비움(Rhizobium)속, 글루코노박터(Gluconobacter)속, 아시네토박터(Acinetobacter)속, 모라셀라(Moraxella)속, 니트로조모나스(Nitrosomonas)속, 아에로모나스(Aeromonas)속, 파라코커스(Paracoccus)속, 바실루스(Bacillus)속, 클로스트리디움(Clostridium)속, 락토바실루스(Lactobacillus)속, 코리네박테리움(Corynebacterium)속, 아르트로박터(Arthrobacter)속, 아크로모박터(Achromobacter)속, 미크로코커스(Micrococcus)속, 마이코박테리움(Mycobacterium)속, 스트렙토코커스 (Streptococcus)속, 스트렙토마이세스(Streptomyces)속, 악티노마이세스(Actinomyces)속, 노카르디아(Nocardia)속, 메틸로박테리움(Methylobacterium)속 등의 각종 세균을 들 수 있다. 또, 상기 세균 이외에, 사카로마이세스(Saccharomyces)속, 칸디다(Candida)속 등의 효모, 또한 각종 곰팡이 등을 숙주로서 들 수 있으며, 이에 제한되지 않는다.Meanwhile, the transformant of the present invention is obtained by introducing the recombinant vector of the present invention into a suitable host. Types of hosts include Escherichia, Pseudomonas, Ralstonia, Alcaligenes, Comamonas, Burkholderia, Genus Agrobacterium, Flabobacterium, Vibrio, Enterobacter, Rhizobium, Gluconobacter, Acinetobacter ) genus, Moraxella genus, Nitrozomonas genus, Aeromonas genus, Paracoccus genus, Bacillus genus, Clostridium genus, Lactobacillus (Lactobacillus), Corynebacterium, Arthrobacter, Achromobacter, Micrococcus, Mycobacterium, Streptococcus ), Streptomyces, Actinomyces, Nocardia, and Methylobacterium. In addition, in addition to the above bacteria, hosts include yeast such as Saccharomyces and Candida, and various fungi, but are not limited thereto.
본 명세서에서 사용되는 용어는 본 발명에서의 기능을 고려하면서 가능한 현재 널리 사용되는 일반적인 용어들을 선택하였으나, 이는 당 분야에 종사하는 기술자의 의도 또는 판례, 새로운 기술의 출현 등에 따라 달라질 수 있다. 또한, 특정한 경우는 출원인이 임의로 선정한 용어도 있으며, 이 경우 해당되는 발명의 설명 부분에서 상세히 그 의미를 기재할 것이다. 따라서 본 발명에서 사용되는 용어는 단순한 용어의 명칭이 아닌, 그 용어가 가지는 의미와 본 발명의 전반에 걸친 내용을 토대로 정의되어야 한다.The terms used in this specification are general terms that are currently widely used as much as possible while considering the function in the present invention, but this may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, etc. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the relevant invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than simply the name of the term.
본 발명은 다양한 변환을 가할 수 있고 여러 가지 실시예를 가질 수 있는 바, 이하 특정 실시예들을 도면에 예시하고 상세한 설명에 상세하게 설명하고자 한다. 그러나, 이는 본 발명을 특정한 실시 형태에 대해 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변환, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다. 본 발명을 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.Since the present invention can be modified in various ways and can have various embodiments, specific embodiments will be illustrated in the drawings and explained in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all transformations, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.
수치 범위는 상기 범위에 정의된 수치를 포함한다. 본 명세서에 걸쳐 주어진 모든 최대의 수치 제한은 낮은 수치 제한이 명확히 쓰여 있는 것처럼 모든 더 낮은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 최소의 수치 제한은 더 높은 수치 제한이 명확히 쓰여 있는 것처럼 모든 더 높은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 수치 제한은 더 좁은 수치 제한이 명확히 쓰여 있는 것처럼, 더 넓은 수치 범위 내의 더 좋은 모든 수치 범위를 포함할 것이다. 본 명세서에 제공된 제목은 다양한 면 또는 전체적으로 명세서의 참조로서, 하기의 구현예를 제한하는 것으로 이해되어서는 안 된다.The numerical range includes the values defined in the range above. Any maximum numerical limit given throughout this specification includes all lower numerical limits as if the lower numerical limit were explicitly written out. Every minimum numerical limit given throughout this specification includes every higher numerical limit as if such higher numerical limit were expressly written out. All numerical limits given throughout this specification will include all better numerical ranges within the broader numerical range, as if the narrower numerical limits were clearly written. The headings provided herein are by reference to the various aspects or of the specification as a whole and should not be construed as limiting the embodiments that follow.
실시예 1 : 우수한 인지질 분해효소를 가지는 박테리아 선별Example 1: Selection of bacteria with excellent phospholipid-degrading enzymes
우수한 인지질 분해효소를 가지는 박테리아를 선별하기 위해 로다민 6G(rhodamine 6G)를 이용하여 효소 활성을 비교하였다. 로다민 6G는 자외선의 350 nm 파장에서 인지질 분해효소의 활성에 따라 빈공간을 만드므로, agar-plate에서 박테리아를 선별하는데 유용하게 활용 가능하다. To select bacteria with excellent phospholipid-degrading enzymes, rhodamine 6G was used to compare enzyme activities. Rhodamine 6G creates empty space according to the activity of phospholipidase at the 350 nm wavelength of ultraviolet light, so it can be usefully used to select bacteria on an agar plate.
인지질 분해효소의 활성을 세포 외에서 agar-plate 방법을 통해 확인하기 위해, Kouker & Jaeger (1987)의 방법을 변형시켜 하기 표 1과 같이 배지를 제작하였다.In order to confirm the activity of phospholipidase outside the cells using the agar-plate method, the method of Kouker & Jaeger (1987) was modified to produce a medium as shown in Table 1 below.
각각의 박테리아에 적합한 액체 배지와 배양 조건에서 키운 후, 600 nm 파장에서 0.1의 흡광도를 나타내는만큼 세포들을 배분하였다. 이후 PBS로 배지를 제거하였으며, 100 μL의 PBS로 풀어주었다. 상기 표 1로 제작한 배지에 적당한 간격으로 깊이 5 mm의 구멍을 내어 준비된 상기 박테리아를 첨가하였다. 사용한 박테리아의 배양 조건에 따라 24시간 동안 배양한 후, 자외선 하에서 인지질의 분해로 색이 변화하여 발생하는 공간의 지름의 길이를 측정하였다(표 2).After growing in a liquid medium and culture conditions suitable for each bacterium, cells were distributed to show an absorbance of 0.1 at a wavelength of 600 nm. Afterwards, the medium was removed with PBS and dissolved in 100 μL of PBS. The bacteria prepared above were added to the medium prepared in Table 1 by making holes with a depth of 5 mm at appropriate intervals. After culturing for 24 hours according to the culture conditions of the bacteria used, the diameter of the space created by the color change due to decomposition of phospholipids under ultraviolet light was measured (Table 2).
그 결과, B. megaterium과 B. licheniformis가 자외선 하에서 발생하는 공간의 지름의 길이가 가장 긴 바, 가장 높은 인지질 분해 활성을 나타내는 것을 확인하였다.As a result, it was confirmed that B. megaterium and B. licheniformis had the longest space diameter under ultraviolet rays and thus exhibited the highest phospholipid decomposition activity.
실시예 2 : 신규 포스포리파아제 유전자 확보와 형질전환Example 2: Obtaining and transforming a new phospholipase gene
상기 실시예 1의 결과를 바탕으로, 하기 표 3과 같이 B. megaterium과 B. licheniformis의 유전자 중 포스포리파아제 A2 활성이 있을 것으로 보이는 유전자 후보군을 선별하였고, 추가로 인지질 분해활성이 뛰어나다고 알려진 Mucilaginibacter gossypiicola, Catenulispora acidiphila로부터 유전자를 선별하였다.Based on the results of Example 1, candidate groups of genes likely to have phospholipase A2 activity were selected among the genes of B. megaterium and B. licheniformis , as shown in Table 3 below, and in addition, Mucilaginibacter , which is known to have excellent phospholipid decomposition activity Genes were selected from gossypiicola and Catenulispora acidiphila .
상기 유전자들을 His-tag가 포함된 박테리아 발현 벡터에 형질전환하였고, DNA 염기서열 분석을 통해 해당 유전자가 삽입되었음을 확인하였다.The above genes were transformed into a bacterial expression vector containing a His-tag, and the insertion of the corresponding gene was confirmed through DNA sequence analysis.
실시예 3 : EPPC 분해 정도로 포스포리파아제 A2 활성 확인Example 3: Confirmation of phospholipase A2 activity by degree of EPPC degradation
실시예 2에서 형질전환된 포스포리파아제 A2 후보군의 효소 활성을 측정하기 위해, 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine (EEPC)의 분해 정도를 확인하였다. To measure the enzyme activity of the phospholipase A2 candidate transformed in Example 2, the degree of degradation of 1,2-α-eleostaroyl-sn-glycero-3-phosphocholine (EEPC) was confirmed.
0.5 mg/mL의 EPPC와 0.01% butylhydroxytoluene(BTH)를 100% 에탄올에 녹인 후 100 μL씩 96 well microtiter 플레이트에 분주하였다. 이 플레이트는 통기가 유용한 클린벤치에서 1차적으로 증발을 시켰고, 이 후 진공 건조기를 이용하여 용매를 완전 증발시켜 EEPC를 플레이트에 코팅하였다. 실험 버퍼로는 150 mM NaCl, 6 mM CaCl2, 1 mM EDTA, 3 mg/mL β을 포함하는 pH 8.0의 10 mM Tris buffer를 이용하였다.0.5 mg/mL of EPPC and 0.01% butylhydroxytoluene (BTH) were dissolved in 100% ethanol and then dispensed at 100 μL each into a 96 well microtiter plate. This plate was first evaporated on a clean bench with good ventilation, and then the solvent was completely evaporated using a vacuum dryer, and EEPC was coated on the plate. As an experimental buffer, 10mM Tris buffer at pH 8.0 containing 150mM NaCl, 6mM CaCl2, 1mM EDTA, and 3mg/mL β was used.
실시예 2의 형질전환 박테리아는 IPTG를 이용해 2시간 동안 효소를 발현하도록 하였다. 박테리아를 파쇄 후 원심분리를 통해 상층액을 수득하였다. 이 때, 상층액의 단백질 농도를 측정하고, 단백질 농도가 모두 같도록 설정하여 실험 버퍼와 교반하였다. 실험을 진행하기 전, 준비한 플레이트를 실험 버퍼를 이용하여 불순물들을 씻어주었고, 기질이 시작 전 평형상태가 되도록 195 μL의 실험 버퍼로 채우고 10분 후 샘플을 5 μL씩 분주하였다. 2시간 동안 30℃에서 반응시킨 후 272 nm에서의 흡광도를 측정하였다. 실험 버퍼의 흡광도를 0으로 맞추고 각각의 샘플에 대해 코팅이 되지 않은 플레이트에 단백질을 분주한 흡광도를 보정하여 측정하였다. The transformed bacteria of Example 2 were allowed to express the enzyme for 2 hours using IPTG. After crushing the bacteria, the supernatant was obtained through centrifugation. At this time, the protein concentration of the supernatant was measured, and all protein concentrations were set to be the same and stirred with the experimental buffer. Before proceeding with the experiment, the prepared plate was washed of impurities using an experimental buffer. It was filled with 195 μL of experimental buffer so that the substrate was in equilibrium before starting, and after 10 minutes, 5 μL of the sample was dispensed. After reacting at 30°C for 2 hours, absorbance was measured at 272 nm. The absorbance of the experimental buffer was set to 0, and for each sample, the absorbance of the protein dispensed onto an uncoated plate was corrected and measured.
그 결과 도 1과 같이 B. megaterium의 EPPC 분해 정도, 즉 포스포리파아제 A2 활성이 가장 높게 나타났으며, 이를 추후 실험에 사용하였다.As a result, as shown in Figure 1, the degree of EPPC degradation, that is, phospholipase A2 activity, of B. megaterium was found to be the highest, and this was used in further experiments.
실시예 4 : 쿠마시 염색법을 통한 Example 4: Via Coomassie staining method B. megateriumB. megaterium 의 포스포리파아제 A2 발현량 확인Confirmation of phospholipase A2 expression level
실시예 3에서 가장 우수한 활성을 나타낸 B. megaterium 유래 포스포리파아제 A2의 발현량을 확인하기 위해, 실시예 3과 같은 발현조건에서 실시예 2에서 사용된 박테리아 발현 벡터에 상기 B. megaterium 유래 포스포리파아제 A2를 형질전환한 두 박테리아를 IPTG를 사용하여 발현하였다. B. megaterium 유래 포스포리파아제 A2의 염기서열 및 아미노산 서열은 각각 하기 표 4에 나타내었다.In order to confirm the expression level of B. megaterium- derived phospholipase A2, which showed the best activity in Example 3, the B. megaterium- derived phospholipase was added to the bacterial expression vector used in Example 2 under the same expression conditions as Example 3. Two bacteria transformed with lipase A2 were expressed using IPTG. The base sequence and amino acid sequence of B. megaterium- derived phospholipase A2 are shown in Table 4 below.
IPTG 발현 조건 외에서 발현되는 단백질과 비교함으로써 원하는 단백질의 발현이 증가하였는지 확인하기 위해, 대조군으로 IPTG를 처리하지 않은 박테리아를 준비하였다.In order to confirm whether the expression of the desired protein increased by comparing it with the protein expressed outside of the IPTG expression conditions, bacteria that were not treated with IPTG were prepared as a control group.
각 박테리아를 파쇄한 후 원심분리기를 이용하여 상층액만을 수득하였다. 각 상층액에 있는 단백질들을 SDS-PAGE를 통해 분리하였으며 분리된 단백질은 쿠마시 염색법을 통해 확인하였다.After crushing each bacteria, only the supernatant was obtained using a centrifuge. Proteins in each supernatant were separated through SDS-PAGE, and the separated proteins were confirmed through Coomassie staining.
그 결과, B. megaterium 유래 포스포리파아제 A2를 형질전환한 샘플에 IPTG를 처리하여 발현시킨 경우, 대조군에서는 발견되지 않은 단백질을 확인하였다(도 2).As a result, when the sample transformed with B. megaterium- derived phospholipase A2 was treated with IPTG and expressed, a protein that was not found in the control group was confirmed (Figure 2).
실시예 5 : 실수유발 중합효소 연쇄 반응 돌연변이화법을 이용해 인지질 분해능이 개선된 포스포리파아제 A2 확보Example 5: Obtaining phospholipase A2 with improved phospholipid decomposition ability using mistake-prone polymerase chain reaction mutagenesis method
실시예 3에서 가장 우수한 활성을 나타낸 B. megaterium 유래 포스포리파아제 A2를 주형 DNA로 사용하여 실수유발 중합효소 연쇄 반응 돌연변이화법을 수행하였다. 상기 실시예 2와 동일한 조건에서 dNTP 중 dATP, dTTP, dGTP, dCTP의 농도를 다르게 해 총 4가지의 중합효소 연쇄 반응의 결과물을 얻었고, 이들을 실시예 2에서 사용된 박테리아 발현 벡터에 형질전환하였다.Phospholipase A2 from B. megaterium , which showed the best activity in Example 3, was used as template DNA to perform mistake-inducing polymerase chain reaction mutagenesis. A total of four polymerase chain reaction results were obtained by varying the concentrations of dATP, dTTP, dGTP, and dCTP among dNTPs under the same conditions as in Example 2, and these were transformed into the bacterial expression vector used in Example 2.
그 결과, 하기 표 5에 나타난 염기서열 및 아미노산 서열을 가진 B. megaterium 유래 포스포리파아제 A2를 포함하는 박테리아가 상대적으로 높은 효소 활성을 나타내었다.As a result, bacteria containing B. megaterium- derived phospholipase A2 with the base and amino acid sequences shown in Table 5 showed relatively high enzyme activity.
포스포리파아제 A2Derived from B. megaterium with improved phospholipid decomposition ability
Phospholipase A2
실시예 6 : 실수유발 중합효소 연쇄 반응 돌연변이화법을 이용해 인지질 분해능이 개선된 포스포리파아제 A2의 활성 확인Example 6: Confirmation of the activity of phospholipase A2 with improved phospholipid decomposition ability using mistake-inducing polymerase chain reaction mutagenesis method
실시예 5에서 확보한 인지질 분해능이 개선된 B. megaterium의 포스포리파아제 A2의 활성을 확인하기 위해, 실시예 3에서 가장 우수한 활성을 나타낸 B. megaterium 유래 포스포리파아제 A2와 상업적으로 사용되고 있는 Novozymes사의 LowP와 비교하여 EPPC 분해 정도를 확인하였다.In order to confirm the activity of B. megaterium phospholipase A2 with improved phospholipid degrading ability obtained in Example 5, B. megaterium- derived phospholipase A2, which showed the best activity in Example 3, and commercially used phospholipase A2 from Novozymes were used. The degree of EPPC decomposition was confirmed by comparison with LowP.
실시예 3과 같이 상기 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2를 실시예 2의 형질전환 박테리아를 이용하여 발현시키고, 박테리아를 파쇄한 후 원심분리를 통해 상층액을 수득하였다. 상층액에 있는 포스포리파아제 A2를 정제하기 위해 4℃에서 Ni-NTA resin와 1시간동안 반응시켰다. 충분히 resin을 씻어준 후 Imidazole이 포함된 버퍼를 이용하여 B. megaterium 유래 포스포리파아제 A2를 정제하였다.As in Example 3, B. megaterium- derived phospholipase A2 with improved phospholipid decomposition ability was expressed using the transformed bacteria of Example 2, and the bacteria were disrupted and the supernatant was obtained through centrifugation. To purify phospholipase A2 in the supernatant, it was reacted with Ni-NTA resin at 4°C for 1 hour. After sufficiently washing the resin, B. megaterium -derived phospholipase A2 was purified using a buffer containing Imidazole.
상기 실시예 3과 같이 활성을 확인하고 그 결과를 효소량에 따른 분당 기질 반응량인 Unit/g로 나타내었다. 그 결과, 실시예 5에서 수득한 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2는 실시예 2에서 수득한 B. megaterium 유래 포스포리파아제 A2보다 높은 활성을 보였으며, 상업적으로 사용되고 있는 Novozymes사의 LowP보다도 활성이 높은 것을 확인하였다(도 3).Activity was confirmed as in Example 3 above, and the results were expressed in Unit/g, which is the amount of substrate reaction per minute according to the amount of enzyme. As a result, B. megaterium- derived phospholipase A2 with improved phospholipid decomposition ability obtained in Example 5 showed higher activity than B. megaterium-derived phospholipase A2 obtained in Example 2, and was commercially used by Novozymes. It was confirmed that the activity was higher than that of LowP (Figure 3).
실시예 7 : 기질량에 따른 인지질 분해능이 개선된 포스포리파아제 A2의 활성 확인Example 7: Confirmation of the activity of phospholipase A2 with improved phospholipid decomposition ability according to the amount of substrate
실시예 6에 기술한 바와 같이, 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2를 정제하고, 실시예 3에서 기질량만을 바꾸어 각각 0.025 μg, 0.05 μg, 0.1 μg, 0.2 μg, 0.4 μg이 포함된 플레이트를 제작하였다. 상기 플레이트를 사용하여 실시예 3에 기술한 바와 같이 EEPC 분해 정도를 측정하였다.As described in Example 6, B. megaterium- derived phospholipase A2 with improved phospholipid decomposition ability was purified, and only the substrate amount was changed in Example 3 to obtain 0.025 μg, 0.05 μg, 0.1 μg, 0.2 μg, and 0.4 μg, respectively. The included plate was produced. The plate was used to measure the degree of EEPC degradation as described in Example 3.
그 결과, 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2는 EEPC가 0.01 μg인 조건에서도 충분한 반응이 일어남을 확인하였다(도 4).As a result, it was confirmed that B. megaterium-derived phospholipase A2, which has improved phospholipid decomposition ability, sufficiently reacts even under conditions where EEPC is 0.01 μg (Figure 4).
실시예 8 : 온도에 따른 인지질 분해능이 개선된 포스포리파아제 A2의 활성 확인Example 8: Confirmation of the activity of phospholipase A2 with improved phospholipid decomposition ability depending on temperature
실시예 6에 기술한 바와 같이, 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2를 정제하고, 최적 반응 온도를 찾기 위해 실시예 3에서 20 내지 40℃로 반응온도를 다르게 하여 효소의 활성을 확인하였다. 플레이트의 온도가 충분히 맞춰질 수 있도록 세척하여 불순물을 제거하거나 화학적 평형을 맞출 때도 반응온도와 동일한 온도의 실험버퍼를 사용하였다. 이후 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2의 활성을 측정하였다.As described in Example 6, phospholipase A2 derived from B. megaterium with improved phospholipid decomposition ability was purified, and the activity of the enzyme was tested by varying the reaction temperature from 20 to 40°C in Example 3 to find the optimal reaction temperature. Confirmed. To ensure that the temperature of the plate was sufficiently adjusted, an experimental buffer with the same temperature as the reaction temperature was used when washing to remove impurities or adjusting chemical equilibrium. Afterwards, the activity of B. megaterium- derived phospholipase A2, which had improved phospholipid decomposition ability, was measured.
그 결과, 37.5℃ 이하에서는 높은 활성을 유지하고, 특히 32.5℃가 최적 온도로서 가장 높은 활성을 나타내었다. 저온에서도 절반 이상의 활성을 유지하는 것을 확인하였다(도 5).As a result, high activity was maintained below 37.5°C, and in particular, 32.5°C was the optimal temperature and showed the highest activity. It was confirmed that more than half of the activity was maintained even at low temperatures (Figure 5).
실시예 9 : 온도에 따른 인지질 분해능이 개선된 포스포리파아제 A2의 잔효성 확인Example 9: Confirmation of residual efficacy of phospholipase A2 with improved phospholipid decomposition ability depending on temperature
실시예 6에 기술한 바와 같이, 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2를 정제하고, 실시예 3에서 반응 전 기질 없이 20 내지 40℃의 온도에서 전보온하는 단계를 추가하였으며, 가장 높은 상대적 활성을 갖는 약 32℃에서 기질과 반응시켰다. As described in Example 6, B. megaterium- derived phospholipase A2 with improved phospholipid decomposition ability was purified, and in Example 3, the step of pre-warming at a temperature of 20 to 40°C without substrate before reaction was added, and the most It was reacted with the substrate at about 32°C with high relative activity.
이후 분해된 기질의 양을 272 nm에서 측정한 결과, 25 내지 35℃에서 전보온한 경우 비교적 활성을 잘 유지하는 것을 확인하였다(도 6).Afterwards, the amount of decomposed substrate was measured at 272 nm, and it was confirmed that the activity was relatively well maintained when preheated at 25 to 35°C (FIG. 6).
실시예 10 : pH에 따른 인지질 분해능이 개선된 포스포리파아제 A2의 활성 확인Example 10: Confirmation of the activity of phospholipase A2 with improved phospholipid decomposition ability according to pH
실시예 6에 기술한 바와 같이, 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2를 정제하고, 실시예 3에서 사용한 실험 버퍼의 pH만을 바꾸어 각각 Phosphate buffer (pH 6.0), Tris buffer (pH 7.0, pH 8.0), Carbonate buffer (pH 9.0)에서 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2의 활성을 측정하였다.As described in Example 6, B. megaterium- derived phospholipase A2 with improved phospholipid decomposition ability was purified, and only the pH of the experimental buffer used in Example 3 was changed to Phosphate buffer (pH 6.0) and Tris buffer (pH 7.0), respectively. , pH 8.0), and carbonate buffer (pH 9.0), the activity of B. megaterium- derived phospholipase A2, which had improved phospholipid decomposition ability, was measured.
그 결과 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2는 pH 8.0에서 가장 높은 활성을 갖는 것이 확인이 되었다(도 7).As a result, it was confirmed that B. megaterium -derived phospholipase A2, which has improved phospholipid decomposition ability, has the highest activity at pH 8.0 (Figure 7).
실시예 11 : pH에 따른 인지질 분해능이 개선된 포스포리파아제 A2의 잔효성 확인Example 11: Confirmation of residual efficacy of phospholipase A2 with improved phospholipid decomposition ability according to pH
실시예 6에 기술한 바와 같이, 인지질 분해능이 개선된 B. megaterium 유래 포스포리파아제 A2를 정제하고, 실시예 10과 같이, 실시예 3에서 사용한 실험 버퍼의 pH를 바꾸되, 기질 없이 전보온 조건만 추가하여 진행하였다. 구체적으로, 같은 양의 포스포리파아제 A2를 Phosphate buffer (pH 6.0), Tris buffer (pH 7.0, pH 8.0), Carbonate buffer (pH 9.0)에서 기질 없이 30분 내지 2시간동안 전보온하였으며, 이후 가장 높은 상대적 활성을 갖는 pH 8.0에서 기질과 반응시켰다. As described in Example 6, B. megaterium- derived phospholipase A2 with improved phospholipid decomposition ability was purified, and as in Example 10, the pH of the experimental buffer used in Example 3 was changed, but prewarmed conditions were used without substrate. We proceeded by adding only. Specifically, the same amount of phospholipase A2 was preheated in Phosphate buffer (pH 6.0), Tris buffer (pH 7.0, pH 8.0), and Carbonate buffer (pH 9.0) without substrate for 30 minutes to 2 hours, and then incubated at the highest It was reacted with the substrate at pH 8.0, which has relative activity.
이후 분해된 기질의 양을 272 nm에서 측정한 결과, pH 8.0에서 전보온한 경우 가장 안정적인 상태를 유지하는 것을 확인하였다(도 8).Afterwards, the amount of decomposed substrate was measured at 272 nm, and it was confirmed that the most stable state was maintained when preheated at pH 8.0 (FIG. 8).
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> KyungHee University Research and Business Foundation <120> Novel phospholipase A2 derived from Bacillus megaterium with enhanced phospholipid hydrolyzation activity <130> GKH21P-0068-KR, DHP21-K216 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 282 <212> DNA <213> Bacillus megaterium <400> 1 atgtctgttc cgaacaaacg gaaagccacg ccctgcctct tccccggcta taaatggtgt 60 ggtcccggct gcagtgggcc cggctgtcct gtaaatgatg tagactgctg ctgtaaatac 120 catgatttat gctatgaaga ttacggatca tgccgctcat gtgatgagca attccttgac 180 tgtctttgct ccaaagcaaa tccgtacagt ttaaaaggaa gacaggcata cgctatgtat 240 acctatatgc ggctgaagtt agctttaaaa caatatgact aa 282 <210> 2 <211> 282 <212> DNA <213> Bacillus megaterium <400> 2 atgtctgttc cgaacaaacg gaaagccgcg ccctgcctct tccccggcta taaatggtgt 60 ggtcccggat gcagtggtcc cggatgtccg gtaaacgatg tagactgctg ctgcaaatac 120 catgatttat gctatgaagg ttacggatca tgccgctcat gtgatgagca atttcttgac 180 tgtctttgct ccaaagcaaa tccgtacagt ttaaaaggaa gacaggcata cgccatgtat 240 acctatatgc gactgaagtt agctttaaaa caatatgact aa 282 <210> 3 <211> 93 <212> PRT <213> Bacillus megaterium <400> 3 Met Ser Val Pro Asn Lys Arg Lys Ala Thr Pro Cys Leu Phe Pro Gly 1 5 10 15 Tyr Lys Trp Cys Gly Pro Gly Cys Ser Gly Pro Gly Cys Pro Val Asn 20 25 30 Asp Val Asp Cys Cys Cys Lys Tyr His Asp Leu Cys Tyr Glu Asp Tyr 35 40 45 Gly Ser Cys Arg Ser Cys Asp Glu Gln Phe Leu Asp Cys Leu Cys Ser 50 55 60 Lys Ala Asn Pro Tyr Ser Leu Lys Gly Arg Gln Ala Tyr Ala Met Tyr 65 70 75 80 Thr Tyr Met Arg Leu Lys Leu Ala Leu Lys Gln Tyr Asp 85 90 <210> 4 <211> 93 <212> PRT <213> Bacillus megaterium <400> 4 Met Ser Val Pro Asn Lys Arg Lys Ala Ala Pro Cys Leu Phe Pro Gly 1 5 10 15 Tyr Lys Trp Cys Gly Pro Gly Cys Ser Gly Pro Gly Cys Pro Val Asn 20 25 30 Asp Val Asp Cys Cys Cys Lys Tyr His Asp Leu Cys Tyr Glu Gly Tyr 35 40 45 Gly Ser Cys Arg Ser Cys Asp Glu Gln Phe Leu Asp Cys Leu Cys Ser 50 55 60 Lys Ala Asn Pro Tyr Ser Leu Lys Gly Arg Gln Ala Tyr Ala Met Tyr 65 70 75 80 Thr Tyr Met Arg Leu Lys Leu Ala Leu Lys Gln Tyr Asp 85 90 <110> KyungHee University Research and Business Foundation <120> Novel phospholipase A2 derived from Bacillus megaterium with enhanced phospholipid hydrolyzation activity <130> GKH21P-0068-KR, DHP21-K216 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 282 <212> DNA <213> Bacillus megaterium <400> 1 atgtctgttc cgaacaaacg gaaagccacg ccctgcctct tccccggcta taaatggtgt 60 ggtcccggct gcagtgggcc cggctgtcct gtaaatgatg tagactgctg ctgtaaatac 120 catgatttat gctatgaaga ttacggatca tgccgctcat gtgatgagca attccttgac 180 tgtctttgct ccaaagcaaa tccgtacagt ttaaaaggaa gacaggcata cgctatgtat 240 acctatatgc ggctgaagtt agctttaaaa caatatgact aa 282 <210> 2 <211> 282 <212> DNA <213> Bacillus megaterium <400> 2 atgtctgttc cgaacaaacg gaaagccgcg ccctgcctct tccccggcta taaatggtgt 60 ggtcccggat gcagtggtcc cggatgtccg gtaaacgatg tagactgctg ctgcaaatac 120 catgatttat gctatgaagg ttacggatca tgccgctcat gtgatgagca atttcttgac 180 tgtctttgct ccaaagcaaa tccgtacagt ttaaaaggaa gacaggcata cgccatgtat 240 acctatatgc gactgaagtt agctttaaaa caatatgact aa 282 <210> 3 <211> 93 <212> PRT <213> Bacillus megaterium <400> 3 Met Ser Val Pro Asn Lys Arg Lys Ala Thr Pro Cys Leu Phe Pro Gly 1 5 10 15 Tyr Lys Trp Cys Gly Pro Gly Cys Ser Gly Pro Gly Cys Pro Val Asn 20 25 30 Asp Val Asp Cys Cys Cys Lys Tyr His Asp Leu Cys Tyr Glu Asp Tyr 35 40 45 Gly Ser Cys Arg Ser Cys Asp Glu Gln Phe Leu Asp Cys Leu Cys Ser 50 55 60 Lys Ala Asn Pro Tyr Ser Leu Lys Gly Arg Gln Ala Tyr Ala Met Tyr 65 70 75 80 Thr Tyr Met Arg Leu Lys Leu Ala Leu Lys Gln Tyr Asp 85 90 <210> 4 <211> 93 <212> PRT <213> Bacillus megaterium <400> 4 Met Ser Val Pro Asn Lys Arg Lys Ala Ala Pro Cys Leu Phe Pro Gly 1 5 10 15 Tyr Lys Trp Cys Gly Pro Gly Cys Ser Gly Pro Gly Cys Pro Val Asn 20 25 30 Asp Val Asp Cys Cys Cys Lys Tyr His Asp Leu Cys Tyr Glu Gly Tyr 35 40 45 Gly Ser Cys Arg Ser Cys Asp Glu Gln Phe Leu Asp Cys Leu Cys Ser 50 55 60 Lys Ala Asn Pro Tyr Ser Leu Lys Gly Arg Gln Ala Tyr Ala Met Tyr 65 70 75 80 Thr Tyr Met Arg Leu Lys Leu Ala Leu Lys Gln Tyr Asp 85 90
Claims (15)
Phospholipase A2 (phospholipase A2) enzyme having phospholipid decomposing activity, consisting of the amino acid sequence of SEQ ID NO: 4 derived from Bacillus megaterium .
상기 서열번호 4의 아미노산 서열로 이루어진 포스포리파아제 A2 효소는 서열번호 3의 10번째 위치에 상응하는 아미노산인 트레오닌이 알라닌으로 치환되고, 47번째 위치에 상응하는 아미노산인 아스파르트산이 글리신으로 치환된 것을 특징으로 하는, 효소.
According to paragraph 1,
The phospholipase A2 enzyme consisting of the amino acid sequence of SEQ ID NO: 4 is characterized in that threonine, the amino acid corresponding to the 10th position of SEQ ID NO: 3, is substituted with alanine, and aspartic acid, the amino acid corresponding to the 47th position, is substituted with glycine. Made with enzymes.
상기 포스포리파아제 A2 효소는 레시틴(lecithin)을 가수분해하여 리소레시틴(lysolecithin)을 생산하는 것을 특징으로 하는, 효소.
According to paragraph 1,
The phospholipase A2 enzyme is an enzyme characterized in that it hydrolyzes lecithin to produce lysolecithin.
상기 포스포리파아제 A2 효소는 32.5℃, pH 8.0에서 최적의 인지질 분해활성을 가지는 것을 특징으로 하는, 효소.
According to paragraph 1,
The phospholipase A2 enzyme is characterized in that it has optimal phospholipid decomposition activity at 32.5°C and pH 8.0.
A polynucleotide consisting of the base sequence of SEQ ID NO: 2, encoding the phospholipase A2 of any one of claims 1 to 4.
An expression vector comprising the polynucleotide of claim 5.
A transformant comprising the expression vector of claim 6.
Phospholipase A2 consisting of the amino acid sequence of SEQ ID NO: 4 derived from Bacillus megaterium ; Or a composition for decomposing phospholipids, comprising as an active ingredient a gene consisting of the base sequence of SEQ ID NO: 2 encoding the same.
A method for producing lysolecithin, comprising the step of treating a substrate with phospholipase A2, which has phospholipid-degrading activity and consists of the amino acid sequence of SEQ ID NO: 4 from Bacillus megaterium .
상기 포스포리파아제 A2는 서열번호 2의 염기서열로 이루어진 유전자를 발현하는 재조합 대장균을 배양하여 수득한 것을 특징으로 하는, 리소레시틴의 생산 방법.
According to clause 9,
A method for producing lysolecithin, wherein the phospholipase A2 is obtained by culturing recombinant E. coli expressing a gene consisting of the base sequence of SEQ ID NO: 2.
상기 단계는 20 내지 37.5℃에서 수행하는 것을 특징으로 하는, 리소레시틴의 생산 방법.
According to clause 9,
A method for producing lysolecithin, characterized in that the step is performed at 20 to 37.5 ° C.
25 내지 35℃에서 기질 없이 상기 포스포리파아제 A2를 첨가하고 30분 내지 2시간 후에, 상기 단계를 수행하는 것을 특징으로 하는, 리소레시틴의 생산 방법.
According to clause 9,
A method for producing lysolecithin, characterized in that the step is performed 30 minutes to 2 hours after adding the phospholipase A2 without a substrate at 25 to 35°C.
상기 단계는 pH 7.5 내지 8.5에서 수행하는 것을 특징으로 하는, 리소레시틴의 생산 방법.
According to clause 9,
A method for producing lysolecithin, characterized in that the step is performed at pH 7.5 to 8.5.
pH 7.5 내지 8.5에서 기질 없이 상기 포스포리파아제 A2를 첨가하고 30분 내지 2시간 후에, 상기 단계를 수행하는 것을 특징으로 하는, 리소레시틴의 생산 방법.
According to clause 9,
A method for producing lysolecithin, characterized in that the step is performed 30 minutes to 2 hours after adding the phospholipase A2 without substrate at pH 7.5 to 8.5.
상기 기질의 최소 농도는 1 μg/mL인 것을 특징으로 하는, 리소레시틴의 생산 방법.
According to clause 9,
A method for producing lysolecithin, characterized in that the minimum concentration of the substrate is 1 μg/mL.
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