Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Properties of a Fibrinolytic Enzyme Secreted by Bacillus amyloliquefaciens RSB34, Isolated from Doenjang

  • Yao, Zhuang (Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University) ;
  • Liu, Xiaoming (Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University) ;
  • Shim, Jae Min (Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University) ;
  • Lee, Kang Wook (Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University) ;
  • Kim, Hyun-Jin (Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University) ;
  • Kim, Jeong Hwan (Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University)
  • Received : 2016.08.17
  • Accepted : 2016.10.06
  • Published : 2017.01.28
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Abstract

Nine bacilli with fibrinolytic activities were isolated from doenjang, a traditional Korean fermented soy food. Among them, RSB34 showed the strongest activity and was identified as Bacillus amyloliquefaciens by 16S rRNA and recA gene sequencing. During growth on LB up to 96 h, RSB34 showed the highest fibrinolytic activity ($83.23mU/{\mu}l$) at 48 h. Three bands of 23, 27, and 42 kDa in size were observed when the culture supernatant was analyzed by SDS-PAGE and 27 and 42 kDa bands by fibrin zymography. The gene encoding the 27 kDa fibrinolytic enzyme AprE34 was cloned by PCR. BLAST analyses confirmed that the gene was a homolog to genes encoding AprE-type proteases. aprE34 was overexpressed in Escherichia coli BL21(DE3) using pET26b(+). Recombinant AprE34 was purified and examined for its properties. The $K_m$ and $V_{max}$ values of recombinant AprE34 were $0.131{\pm}0.026mM$ and $16.551{\pm}0.316{\mu}M/l/min$, respectively, when measured using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. aprE34 was overexpressed in B. subtilis WB600 using pHY300PLK. B. subtilis transformants harboring pHYRSB34 (pHY300PLK with aprE34) showed higher fibrinolytic activity than B. amyloliquefaciens RSB34.

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References

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