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

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.

Cloning and overexpression of aprE3-17 encoding the major fibrinolytic protease of Bacillus licheniformis CH 3-17

“…This pattern was different from that reported for a fibrinolytic enzyme from B. subtilis, by which the Bβ chain was degraded first [16]. The pattern was also different from that reported for recombinant subtilisin DJ-4, by which the γ-chain was degraded [8]. The detailed degradation pattern for fibrinogen could be different among similar fibrinolytic proteins, and thus each enzyme should be individually characterized for this property.…”

“…This phenomenon, so-called "a binding mode," was caused by either the tight binding of enzyme to fibrin in the gel or a high pI value of the enzyme. The same phenomenon was observed for AprE51 [7] and other fibrinolytic enzymes [1,8,15]. Since the calculated pI value of AprE3-17 (6.65) is not high enough to cause this phenomenon, the binding mode might be caused by binding of AprE3-17 to fibrin in the gel.…”

“…B. subtilis secretes several proteases into the culture medium, including alkaline protease (subtilisin, encoded by apr), neutral protease (encoded by npr), bacillopeptidase F (encoded by bpr), Epr (extracellular protease, encoded by epr), Mpr (extracellular metalloprotease, encoded by mpr), and Vpr (extracellular serine protease, encoded by vpr). Among them, subtilisin and neutral protease are the most important enzymes and are responsible for > 90% of the total extracellular protease activity [1,8]. In our previous studies, aprE homologs were cloned from B. subtilis CH3-5 and B. amyloliquefaciens CH51, respectively; both were also members of the six isolates [7,9].…”

Genetic identification and expression optimization of a novel protease HapR from Bacillus velezensis

“…This result suggested that the gene hapR of B. velezensis WH-7 was the key protease gene for degrading proteins in tobacco. Previous studies demonstrated that there are different proteases in Bacillus species, such as AprE ( Hata et al, 2001 ; Sareen et al, 2005 ; Shi et al, 2019 ), subtilisin DJ-4 (AY627764) ( Choi et al, 2004 ), DFE (DQ132806) ( Peng et al, 2003 ), BsfA (JN392072.1) ( Hu et al, 2019 ), BAF1 (FJ517584.1) ( Agrebi et al, 2009 ). Herein, the protease HapR was found to be the key protease in B. velezensis for the first time.…”

Secretory Expression of Nattokinase from Bacillus subtilis YF38 in Escherichia coli

“…Production of fibrinolytic enzymes including nattokinase by expression of them in E. coli has been previously reported [10][11][12], but in most cases they were produced as inactive inclusion bodies or insoluble aggregates, and fibrinolytic active enzymes could only be obtained by renaturation from the inclusion bodies [10,11] or by the IMPACT (Intein Mediated Purification with an Affinity Chintin-binding Tag) system from the insoluble recombinant protein [12].…”