Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Characterization of a Fibrinolytic Enzyme Secreted by Bacillus amyloliquefaciens CB1 and Its Gene Cloning

  • Heo, Kyeong (Division of Applied Life Science BK21, Graduate School, Gyeongsang National University) ;
  • Cho, Kye Man (Department of Food Science, Gyeongnam National University of Science and Technology) ;
  • Lee, Chang Kwon (Mong-Go Foods Co., Ltd.) ;
  • Kim, Gyoung Min (Namhae Garlic Research Institute) ;
  • Shin, Jung-Hye (Namhae Garlic Research Institute) ;
  • Kim, Jong Sang (School of Applied Biosciences and Food Science and Biotechnology, Kyungpook National University) ;
  • Kim, Jeong Hwan (Division of Applied Life Science BK21, Graduate School, Gyeongsang National University)
  • Received : 2013.02.26
  • Accepted : 2013.03.30
  • Published : 2013.07.28
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Abstract

Bacillus amyloliquefaciens CB1 was isolated from cheonggukjang, a Korean fermented soy food. B. amyloliquefaciens CB1 secretes proteases with fibrinolytic activities. A gene homologous to aprE of Bacillus subtilis, aprECB1, was cloned from B. amyloliquefaciens CB1, and DNA sequencing showed that aprECB1 can encode a prepro-type serine protease consisting of 382 amino acids. When aprECB1 was introduced into B. subtilis WB600 using an E. coli-Bacillus shuttle vector, pHY300PLK, transformants showed fibrinolytic activity and produced a 28 kDa protein, the size expected for the mature enzyme. The 28 kDa fibrinolytic enzyme was purified from the culture supernatant of B. subtilis WB600 transformant. AprECB1 was completely inhibited by phenylmethylsulfonyl fluoride and almost completely inhibited by EDTA and EGTA, indicating that it is a serine metalloprotease. AprECB1 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, a known substrate for ${\alpha}$-chymotrypsin. $A{\alpha}$ and $B{\beta}$ chains of fibrinogen were quickly degraded by AprECB1, but the ${\gamma}$-chain was resistant.

Keywords

References

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