Cloning, expression, and fibrin (ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. DJ-4

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Title
Cloning, expression, and fibrin (ogen)olytic properties of a subtilisin DJ-4 gene from Bacillus sp. DJ-4
Author(s)
Nack Shick Choi; Kyu Tae Chang; P J Maeng; Seung Ho Kim
Bibliographic Citation
FEMS Microbiology Letters, vol. 236, no. 2, pp. 325-331
Publication Year
2004
Abstract
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.
Keyword
BacillusFibrinolytic enzymePlasminogen activatorRecombinant subtilisin DJ-4
ISSN
0378-1097
Publisher
Oxford Univ Press
DOI
http://dx.doi.org/10.1016/j.femsle.2004.06.006
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
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