2017 ; Vol.27-1: 9~18
|Author||Zhuang Yao, Xiaoming Liu, Jae Min Shim, Kang Wook Lee, Hyun-Jin Kim, Jeong Hwan Kim|
|Place of duty||Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University, Jinju 52828, Republic of Korea|
|Title||Properties of a Fibrinolytic Enzyme Secreted by Bacillus amyloliquefaciens RSB34, Isolated from Doenjang|
J. Microbiol. Biotechnol.2017 ;
|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 (83.23 mU/μl) at 48 h. Three bands of 23,
27, and 42 kDa in size were observed when the culture supernatant was analyzed by SDSPAGE
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 Km and Vmax values of recombinant AprE34 were 0.131 ± 0.026 mM and
16.551 ± 0.316 μM/l/min, respectively, when measured using an artificial substrate, Nsuccinyl-
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.|
|Key_word||Bacillus amyloliquefancies, fibrinolytic enzymes, doenjang, gene cloning|
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