Characterization of a Fibrinolytic Enzyme Secreted by Bacillus amyloliquefaciens CB1 and Its Gene Cloning

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 α-chymotrypsin. Aα and Bβ
chains of fibrinogen were quickly degraded by AprECB1, but the γ-chain was resistant.

Key words: Fibrinolytic enzyme, protease, Bacillus amyloliquefaciens