Open AccessCloning, expression, and fibrin (ogen)olytic properties of a subtilisin DJ‐4 gene from Bacillus sp. DJ‐4
Author(s) -
Choi NackShick,
Chang KyuTae,
Jae Maeng Pil,
Kim SeungHo
Publication year - 2004
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2004.tb09665.x
Subject(s) - subtilisin , microbiology and biotechnology , chemistry , plasmin , biochemistry , nattokinase , enzyme , plasminogen activator , biology , genetics , fermentation
Previously, we purified a strong fibrinolytic enzyme (subtilisin DJ‐4) from Bacillus sp. DJ‐4 and characterized its enzymatic activity. Here, we cloned the gene subtilisin DJ‐4, and determines its nucleotide sequence, which showed 97% identity with subtilisin BPN′ from B. amyloliquefacens . Recombinant full‐subtilisin DJ‐4 (rf‐subDJ‐4) and mature‐subtilisin DJ‐4 (rm‐subDJ‐4) were expressed using a pET29 vector system, and their fibrin (ogen)olytic and plasminogen activator activities were studied. rf‐subDJ‐4 was found to have a higher stability to heat (60 °C) and to acidic conditions (pH 3.0–4.0) than the native subtilisin DJ‐4 of Bacillus sp. DJ‐4. The plasminogen activator activity of rf‐subDJ‐4 was 2.75 times greater than that of plasmin on a molar basis. And its specific activity (F/C, the ratio of fibrinolytic activity to caseinolytic activity) was 2.67 and 3.97 times higher than those of subtilisin BPN′ and subtilisin Carlsberg, respectively. rf‐subDJ‐4 rapidly hydrolyzed the Aα‐, Bβ‐, and γ‐chains of fibrinogen within 5 min. But, unlike subtilisin BPN′ at a very low concentration (50 ng), the γ‐chain was not cleaved. On the other hand, rm‐subDJ‐4 did not show enzyme activity.