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

FEMS Microbiology Letters
Nack-Shick ChoiSeung-Ho Kim

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 degrees 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 Aalpha-, Bbeta-, and gamma-chains of fibrinogen within 5 min. But, unlike subtilisin BPN' at a very low concentration (50 ng), the gamma-chain was not cleaved. On the other hand, rm-subDJ-4 did not show enzyme activity.

Citations

Oct 8, 2005·Applied Microbiology and Biotechnology·Yong PengYizheng Zhang
Apr 27, 2007·Applied Microbiology and Biotechnology·Qiuhong NiuKeqin Zhang
Oct 24, 2007·Molecular Biotechnology·Xiaobo LiangLiandong Huan
Nov 10, 2011·Molecular Biotechnology·Younes GhasemiAbdollah Ghasemian
Jun 11, 2011·BMC Biotechnology·Ekkarat PhrommaoMontarop Yamabhai
Feb 24, 2006·Bioscience, Biotechnology, and Biochemistry·Endang SetyoriniKenji Aoki
Jan 14, 2011·Bioscience, Biotechnology, and Biochemistry·Shinji TakenakaKenji Aoki

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