Characterization of AprE176, a Fibrinolytic Enzyme from Bacillus subtilis HK176

Abstract

Bacillus subtilis HK176 with high fibrinolytic activity was isolated from cheonggukjang, a Korean fermented soyfood. A gene, aprE176, encoding the major fibrinolytic enzyme was cloned from B. subtilis HK176 and overexpressed in E. coli BL21(DE3) using plasmid pET26b(+). The specific activity of purified AprE176 was 216.8 ± 5.4 plasmin unit/mg protein and the optimum pH and temperature were pH 8.0 and 40°C, respectively. Error-prone PCR was performed for aprE176, and the PCR products were introduced into E. coli BL21(DE3) after ligation with pET26b(+). Mutants showing enhanced fibrinolytic activities were screened first using skim-milk plates and then fibrin plates. Among the mutants, M179 showed the highest activity on a fibrin plate and it had one amino acid substitution (A176T). The specific activity of M179 was 2.2-fold higher than that of the wild-type enzyme, but the catalytic efficiency (kcat/Km) of M179 was not different from the wild-type enzyme owing to reduced substrate affinity. Interestingly, M179 showed increased thermostability. M179 retained 36% of activity after 5 h at 45°C, whereas AprE176 retained only 11%. Molecular modeling analysis suggested that the 176(th) residue of M179, threonine, was located near the cation-binding site compared with the wild type. This probably caused tight binding of M179 with Ca(2+), which increased the thermostability of M179.

Characterization of a Fibrinolytic Enzyme Secreted by Bacillus velezensis BS2 Isolated from Sea Squirt Jeotgal

“…Considering the results, AprEBS2 has moderate heat stability. The optimum temperature for AprEBS2 is similar to that for AprEs from other Bacillus strains such as B. subtilis HK176 [31] and B. amyloliquefaciens RSB34 [14], but lower than that for Bacillus sp. strain CK11-4 (70 o C) [32].…”

Properties of a fibrinolytic enzyme secreted by Bacillus subtilis JS2 isolated from saeu (small shrimp) jeotgal

“…2). aprEJS2 showed 99% nucleotide sequence identity to other gene from Bacillus amyloliquefaciens MJ5-41 (JF739176, 1135/1149) [17], Time (h) and 98% identities to genes from B. amyloliquefaciens CH51 (EU414203, 1129/1149) [16], and B. subtilis HK176 (KJ572414, 1129/1149) [19]. AprEJS2 showed high similarities with other fibrinolytic enzymes: 99.4% with AprE5-41 from B. amyloliquefaciens MJ5-41 (AEE81297), and AprE51 from B. amyloliquefaciens CH51 (ACA34903), and 98.4% with AprE176 from B. subtilis HK176 (AHN52401).…”

Properties of Antimicrobial Substances Produced by Bacillus amyloliquefaciens CJW15 and Bacillus amyloliquefaciens SSD8

“…Fibrinolytic activities of B. amyloliquefaciens CJW15 and SSD8 increased and then decreased as the growth continued, which were quite different from the control, B. subtilis HK176 [7], a strain with strong fibrinolytic activity and its fibrinolytic activity increased continuously with incubation time (Fig. 3).…”

“…Ammonium sulfate powder was very slowly added to each 200 ml FS from SSD8 and Fibrinolytic activity of B. amyloliquefaciens CJW15 and SSD8 B. amyloliquefaciens CJW15 and SSD8 were cultivated in TSB (1%, v/v) for 84 h 37 o C. At 12 h intervals, 5 ml of culture was taken out and centrifuged at 4,000 × g for 10 min. FS was used as the sample for the fibrinolytic activity measurement by fibrin plate method [7]. Fibrinogen (Sigma) solution (0.5%, w/v) in 10 ml of 1 M phosphate-buffered saline (PBS) was mixed with an equal volume of 2% (w/v) agarose solution and then poured onto a plate, which previously evenly distributed with 0.1 ml of thrombin solution (100 NIH units/ml, Sigma).…”

“…The size was converted into plasmin units (U) by comparing it to those from plasmin standards. A standard curve showing the relationship was obtained [7]. Supernatant from B. subtilis HK 176 was used as a positive control [7].…”