Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification and Partial Amino Acid Sequence of a Bacteriocin Produced by Lactococcus, sp. HY449

Lactococcus sp. HY449가 생산한 Bacterisocin의 정제

  • 오세종 ((주)한국야쿠르트 중앙연구소) ;
  • 이상준 (청강문화산업대학 건강식품과학과) ;
  • 김경태 ((주)한국야쿠르트 중앙연구소) ;
  • 김상교 ((주)한국야쿠르트 중앙연구소) ;
  • 박연희 (아주대학교 생물공학과) ;
  • 백영진 ((주)한국야쿠르트 중앙연구소)
  • Published : 2001.09.01
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Abstract

A bactriocin produced by Lactrococcus sp. HY449 was purified by sequential purfication steps such as n-propanol-acetone precipitation ion -exchange chromatography using CM-Sequential CL6B. gel filtration chromatography using Sephacry HR100 and reverse-phase chromatography using pro RPC HR 5/10. Reverse-phase chromatography the final step of the purfication yielded a single symmetrical peak of bacteriocin activity The purification resulted in final yield of 3.25% and 413.35 fold increase of the specific activity of bacteriocin. The active fraction from reverse-phase chromatography was used for N-terminal amino acid analysis . The purified bacteriocin contained isoleucine, leucine, methionine, and glycine at but N-terminal end no aromatic amino acids. Calculation of the number of amino acid residues in the bacteriocin revealed that it is consisted of 32 residues assuming the molecular weight of bacteriocin to be about 3.6kDa. Edman degrandation elucidated amino acid residues of the first four of the N-terminus to be $NH_2$-Ile-Leu-Pro-GIn.

Lactrococcus sp. HY449균줄르 M17-glucose broth에 배양하여 배양 상등액으로부터 propanol-actone 침전 ion-exchange chromatography gel-filtration chromatography 및 reverse-phase chroamtography 등을 통하여 비활성 25,600,000 BU/mg 인 순수한 bacteriocin 을 정제하였다. 정제 과정 주에서 ion-exchange chromatography 단계에 서는 35.3%의회수율이 7.3%로 감소하였다. Reverse-Phase chromatography에선 3.3%의 회수율을 보였고 활성도는 413.5배로 증가하였다. Tricine-SDS 전기영도 결과 bacteriocin 은 단일 밴드로 나타났으며, N-말단 아미노산 서열 분석을 수행한 결과 $NH_2$-IIe-Leu-Pro-GIn로 확인되었다. 아미노산조정 분석결과를 바탕으로 분자량을 예측한 결과 본 bacteriocin은 32개의 아미노산으로 이루어져 있으며 분자량은 3.6kDa인 것으로 추정되었다.

Keywords

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