Properties of a Fibrinolytic Enzyme Secreted by Bacillus amyloliquefaciens RSB34, Isolated from Doenjang.

Nine bacilli with fibrinolytic activities were isolated from Doenjang , a traditional Korean fermented soy food. Among them, RSB 34 showed the strongest activity and identified as Bacillus amyloliquefaciens by 16S rRNA and recA genes 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 culture supernatant was analyzed by SDS-PAGE and 27 and 42 kDa bands by fibrin zymography. A gene encoding the 27 kDa major fibrinolytic enzyme, AprE34, was cloned by PCR. BLAST searches 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 the properties. Km and Vmax values of recombinant AprE34 were 0.131±0.026 mM and 16.551±0.316 μM/l/min, respectively, when measured by 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.