A simple and rapid screening method for microorganisms with phospholipase $A_1$, $A_2$ and C activities using agar plate and gas chromatography (GC) method was successfully carried out. In agar plate method, soy bean lecithin and taurocholic acid were used as carbon source and emulsifier, respectively. In this agar plate method, microorganisms with phospholipase $A_1$ and $A_2$ or C activity produce a halo around the colony and two kinds(A's and C) of microorganisms are clearly distinguished by turbidity of the halo. Microorganisms with phospholipase $A_1$ and $A_2$ activity is simply distinguished by GC using a synthetic phospholipid containing different fatty acid at sn-1 and sn-2 position.
Using this screen method, a bacterium which has phospholipase $A_1$ activity was isolated from soil. This microorganism was aerobic, motile, oxidase negative and had flagella. The G + C content of the DNA base was 58.1 mol%. The major isoprenoids of cell wall were Q-8 and MK-8. The main cellular fatty acids were saturated straight chain(n-16) and cyclic(17:△) fatty acid. Based on their morphological, physiological and chemotaxonomic characteristics, this strain was placed in the genus Serratia and named MK1. Nutritional factors affecting enzyme production were explored. Xylose and ammonium sulfate were found to be the best carbon and nitrogen sources, respectively. Ferrous ion exerted a positive effect on enzyme production considerably. The optimal pH and temperature for phospholipase $A_1$ production were determined to be about 7.0 and 30℃, respectively.
A novel type of extracellular phospholipase $A_1$ was isolated from Serratia sp. MK1 and purified to homogeneity by ammonium sulfate precipitation, anion exchange chromatography and gel filtration. The purified enzyme was a monomer with a molecular mass of about 43,000 Da. This enzyme showed the highest lipolytic activity toward phosphatidylserine among the phosphoglycerides tested, and preferentially catalyzed the hydrolysis of ester bond in phosphatidic acid to lyso-phosphatidic acid. The enzyme activity was completely inhibited by addition of chelating agent such as EDTA and inhibited enzyme activity was fully recovered by the presence of $Ca^{2+}$. It implies that the enzyme requires $Ca^{2+}$ for their activity. The enzyme was stable up to 70℃ when incubated for 1hr at pH 8.5, and the optimal pH and temperature were 8.5 and 50℃, respectively.
A gene coding for a extracellular phospholipase $A_1$ of Serratia sp. MK1 was cloned into E. coli DH5α by inserting EcoRI generated DNA fragment into the pUC19. One colony possessing phospholipase $A_1$ activity on the selective medium was isolated by screening the constructed Serratia sp. MK1 genomic library.
The complete nucleotide sequence of the phospholipase $A_1$ gene was identified.
The sequenced gene consisted of two open reading frame, 966 and 705 bp long.
One of the gene product was identified as a phospholipase $A_1$ and deduced amino acid sequence of phospholipase $A_1$ showed high overall homology to the phospholipase $A_1$ from Serratia liquefaciens, lipase from Serratia proteomaculans and extracellular nuclease from Serratia spp. Second open reading frame showed high homology to PhlB gene among the two open reading frame of phospholipase $A_1$ operon from Serratia liquefaciens.
Hydrolysis of phospholipid using phospholipase $A_1$ from Serratia sp. MK1 has been studied in an organic-aqueous two phase system and emulsion system. In two phase system, hydrolysis product of phospholipid, lyso-phospholipid, was accumulated in organic phase, but in emulsion system, lyso-phospholipid was hydrolyzed to phosphatidic acid. Considering this result, two phase system was more appropriate than emulsion system for production of lyso-phospholipid.
Among the organic solvents tested, butyl acetate was the most suitable solvent for this purpose. The optimal substrate concentration in butyl acetate was 10%. When the reaction was carried out under the optimized condition, phospholipid was hydrolyzed completely to the lysophospholipid and lyso-phospholipid was easily separated from the reaction system.
1. 포스포리파제 $A_1$, $A_2$, C역가를 가진 균주를 탐색하기위한 간단하고도 신속한 탐색방법을 한천배지와 기체 크로마토그래피를 이용하여 개발하였다. 한천배지 방법에서는 콩의 인산지방질과 타우로콜린산을 탄소원과 계면활성제로 각각 사용하였다. 이 배지에서는 포스포리파제 $A_1$과 $A_2$ 또는 C의 역가를 가진 균주를 쉽게 분리할 수 있었으며 포스포리파제 $A_1$과 $A_2$의 역가를 가진균주는 각각 sn-1 과 sn-2 위치에 각각 다른 지방산이 붙어있는 합성 인산지방질을 이용하여 기체 크로마토그래피를 이용하여 쉽게 분리할 수 있었다.
2. 위의 탐색 방법을 이용하여 포스포리파제 $A_1$의 역가를 가진 균주를 토양으로부터 분리했다. 이 미생물은 이동성이 있는 호기성 균주로써 옥시다제 음성이며 편모를 가지고 있었다. DNA의 G+C 함량은 58.1mol\%였으며 세포내 주 isoprenoid는 Q-8과 MK-8 이었다. 주된 세포내 지방산은 직쇄형 포화 지방산(n-16)과 고리형 지방산 (17:△)이였다. 이와같은 형태학적, 생리학적 특성에 의해 분리한 균주를 Serratia 속에 속하는 미생물로 동정하였으며 Serratia sp. MK1 이라 명명하였다.
3. 효소를 생산하기 위한 생산조건에 있어서는 Xylose와 ammonium sulfate가 가장 좋은 탄소 및 질소원이었으며 철 이온은 효소생산에 좋은 영향을 미쳤다. 또한 효소생산을 위한 배지의 최적 pH 및 온도는 각각 7.0 및 30℃였다.
4. Serratia sp. MK1이 생산하는 포스포리파제 $A_1$을 ammonium sulfate precipitation, 이온교환수지 및 gel filtration chromatography를 이용하여 정제하였다. 정제한 효소는 분자량이 약 43,000인 subunit이 하나인 단백질이었다. 정제한 효소는 각종 인산지방질중 phosphatidylserine에 가장 큰 기질 특이성을 보였다. 또한 효소의 역가에는 칼슘이온이 절대적으로 필요했다. 효소의 최적 반응 조건은 pH 8.5, 55℃였으며 효소의 온도에 대한 안정성은 70℃에서 반감기가 1시간이었다.
5. Serratia sp. MK1이 생산하는 포스포리파제$A_1$을 coding하는 유전자를 분리하여 염기서열을 결정하였다. 이때 포스포리파제 $A_1$ 유전자는 두개의 open reading frame으로 구성되어 있었고 각각의 크기는 966과 705bp였다. 이들 두개의 open reading frame은 각각 PLA와 PhlB로 명명하였으며 이중 PLA가 포스포라파제 $A_1$을 coding하는 유전자였다. 염기서열로부터 추정한 아미노산 서열로부터 Serratia sp. MK1이 생산하는 포스포리파제 $A_1$은 Serratia 속이 생산하는 리파제, nuclease, 포스포리파제 $A_1$과 높은 homology를 보였다.
6. 각각 이상계와 에멀젼계에서 Serratia sp. MK1이 생산하는 포스포리파제 $A_1$을 이용하여 인산지방질을 분해하였다. 이때 이상계에서는 라이소 인산지방질이 반응조내에 축적되었으나 에멀젼계에서는 생성된 라이소 인산지방질도 가수분해되어 버림을 관찰할 수 있었다, 위의 결과로부터 라이소 인산지방질을 생산하기 위한 반응계로 이상계를 선택하였다. 이상계에서 사용될 각종 유기용매를 시험해 본 결과 butyl acetate 가 가장 적절한 유기용매임을 알 수 있었고 유기용매층에서의 기질의 농도를 20%(w/v)까지 높여도 대부분의 인산지방질이 라이소 인산지방질로 가수분해되었다.
