Lactobacillus acidophilus GP4A 박테리오신의 정제, Bacteriolytic 작용 및 생산 관련 Plasmid의 선별

Purification, Bacteriolytic Action and Plasmid Isolation of Acidocin 4A Produced by Lactobacillus acidophilus GP4A

Lactobacillus acidophilus CP4A 균주가 생산하는 acidocin 4A를 정제하고자 ammonium sulphate 침전법 , Octyl-sepharose CL-4B column chromatography, $C_{18}$ Sep-Pak cartridge, $C_{18}$ RP HPLC, HPLC gel filtration을 실시하였고 tricine SDS-PAGE를 통해 약 4.1 kDa의 박테리오신임을 확인하였다. Acidocin 4A의 항균작용 기작을 L. delbrueckii subsp. lactis ATCC 4797을 대상으로 TEM을 이용해 관찰한 결과 세포벽이 해리되고 세포벽사이로 세포내용물이 용출되어 궁극적으로 cell lysis가 일어나는 bacteriolytic 현상을 확인하였다. 또한, acidocin 4A의 생산에 관련된 유전자의 존재 위치를 파악하고자 EtBr을 이용한 curing방법을 실시하였으며 그 결과 약 20 kb 크기의 plasmid에 acidocin 4A 생산과 자체 면역에 관련된 유전자가 존재함을 알 수 있었다.

Acidocin 4A produced by Lactobacillus acidophilus GP4A was purified to homogeneity by ammonium sulfate precipitation and sequential chromatographies containing Octyl sepharose CL-4B column, $C_{18}$ Sep-Pak Cartridge, $C_{18}$ RP HPLC and HPLC gel filtration. Tricine SDS-PACE resulted in a single band with estimated molecular mass of 4.1 kDa corresponding to the polypeptide weight marker. Electron microscopy of acidocin-treated indicator cells(L. delbrueckii subsp. lactis ATCC 4797) confirmed that acidocin 4A presented bacteriolytic effect, resulting in cell lysis. Curing trial using ethidium bromide (EtBr) was carried out to examine whether acidocin 4A determinant was encoded either by chromosome or on plasmid. The plasmid designated as pLA4A, being about 20 kb in size, was responsible for acidocin 4A production and immunity to host cells.