Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Enhanced Activity of Cytidine Deaminase by Gene Family Shuffling.

Gene Family Shuffling을 이용한 Cytidine Deaminase 활성 증가

  • Hong, Sik (Department of Microbiology, Kyungpook National University) ;
  • Kim, Kyung-Dong (Department of Microbiology, Kyungpook National University) ;
  • Song, Bang-Ho (Department of Biology Education, Kyungpook National University) ;
  • Jung, Kyung-Hwa (Genomics Laboratory, Amicogen Inc.) ;
  • Kim, Sa-Yeol (Department of Microbiology, Kyungpook National University)
  • 홍식 (경북대학교 미생물학과) ;
  • 김경동 (경북대학교 미생물학과) ;
  • 송방호 (경북대학교 생물교육과) ;
  • 정경화 (아미코젠(주) 제노믹스연구실) ;
  • 김사열 (경북대학교 미생물학과)
  • Published : 2002.12.01
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Abstract

A family shuffling associating PCR-based and in vitro recombination and expression in Escherichia coli cdd mutant was carried out. Two cdd genes encoding cytidine deaminases (CDase) from thermophilic Bacillus caldolyticus and B. stearothermophilus were shuffled. Around 150 viable mutant colonies screened on AB minimal medium without uracil by E. coli cdd complementation were selected for cytidine deaminase assay and 4 candidates (SH1067, SH1077, SH1086, and SH1118) were chosen for the detailed study. The nucleotide sequence analyses of 4 selected mutants revealed that they have several point mutations and recombinations. Surprisingly, the SH 1067 showed 770 fold more specific CDase activity at $80^{\circ}C$ than that of T101 from parental B. stearothermophilus.

PCR방법을 기본으로 한 in vitro recombination과 대장균 cdd 돌연변이주에서의 발현을 통하여 family shuffling이 수행되었다. 고온성 Bacillus caldolyticu와 B. stearothermophilus 유래의 시티딘 디아미나제을 코드하는 cdd 유전자를 shuffling하였다. 이것을 대장균 cdd 돌연변이주에 형질전환 시킨 후 uraci이 없는 AB배지에서의 생존을 통하여 150개의 돌연변이 균주를 얻을 수 있었으며, 그 중 연구를 위하여 4주(SH1067, SH1077, SH1086, 및 SH1118)를 선택하였다. 선택한 4주의 염기서열을 분석한 결과, 수 회의 point mutation과 recombination이 각각 일어났음을 확인 할 수 있었다. 특히 SH1067의 경우,$ 80^{\circ}C$에서 B. stearothemophilus 에서 유래한 7101의 시티딘 디아미나제 활성과 비교하여 770배 이상의 증가를 보여주었다.

Keywords

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