title: Characterization of a fibrinolytic enzyme secreted by Bacillus amyloliquefaciens CB1 and its gene cloning.
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 α-chymotrypsin. Aα and Bβ chains of fibrinogen were quickly degraded by AprECB1, but the γ-chain was resistant.
authors.affiliation: Division of Applied Life Science BK21, Graduate School, Gyeongsang National University, Jinju 660-701, Republic of Korea.
authors.forename: Kyeong
authors.initials: K
authors.lastname: Heo
authors.forename: Kye Man
authors.initials: KM
authors.lastname: Cho
authors.forename: Chang Kwon
authors.initials: CK
authors.lastname: Lee
authors.forename: Gyoung Min
authors.initials: GM
authors.lastname: Kim
authors.forename: Jung-Hye
authors.initials: JH
authors.lastname: Shin
authors.forename: Jong Sang
authors.initials: JS
authors.lastname: Kim
authors.forename: Jeong Hwan
authors.initials: JH
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004492:Edetic Acid / Q000378:metabolism; D004533:Egtazic Acid; D004791:Enzyme Inhibitors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005341:Fibrinolysin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005516:Food Microbiology; D005822:Genetic Vectors; D007723:Korea; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D004533:Egtazic Acid; D010664:Phenylmethylsulfonyl Fluoride; D004492:Edetic Acid; D057057:Serine Proteases; D005341:Fibrinolysin