Biochemical analysis of a fibrinolytic enzyme purified from Bacillus subtilis strain A1

Yeo, Won Sik; Seo, Min Jeong; Kim, Min Jeong; Lee, Hye Hyeon; Kang, Byoung Won; Park, Jeong Uck; Choi, Yung Hyun; Jeong, Yong Kee

doi:10.1007/s12275-011-1165-3

Cited by 11 publications

(3 citation statements)

References 21 publications

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“…4A and 4B). The optimal temperature for FSP3 coincided with that for FP28 [47] and Bacillokinase II [61] and was lower than that reported for AJ (85°C) [6], but higher than that reported for subtilisin DFE (48°C) [46], N-V protease (45°C) [62], CSP(40°C) [35], and MEF (30°C) [18]. As shown in Fig.…”

Section: Effects Of Ph and Temperaturesupporting

confidence: 66%

Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3

Cheng¹,

He²,

Sun³

et al. 2015

Journal of Microbiology and Biotechnology

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A novel proteolytic enzyme with fibrinolytic activity, FSP3, was purified from the recently isolated Streptomyces sp. P3, which is a novel bacterial strain isolated from soil. FSP3 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange, and gel filtration. FSP3 is considered to be a single peptide chain with a molecular mass of 44 kDa. The maximum activity of the enzyme was observed at 50°C and pH 6.5, and the enzyme was stable between pH 6 and 8 and below 40°C. In a fibrin plate assay, FSP3 showed more potent fibrinolytic activity than urokinase, which is a clinical thrombolytic agent acting as a plasminogen activitor. The activity was strongly inhibited by the serine protease inhibitor PMSF, indicating that it is a serine protease. Additionally, metal ions showed different effects on the activity. It was significantly suppressed by Mg(2+) and Ca(2+) and completely inhibited by Cu(2+), but slightly enhanced by Fe(2+). According to LC-MS/MS results, its partial amino acid sequences are significantly dissimilar from those of previously reported fibrinolytic enzymes. The sequence of a DNA fragment encoding FSP3 contained an open reading frame of 1287 base pairs encoding 428 amino acids. FSP3 is a bifunctional enzyme in nature. It hydrolyzes the fibrin directly and activates plasminogen, which may reduce the occurrence of side effects. These results suggest that FSP3 is a novel serine protease with potential applications in thrombolytic therapy.

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Section: Effects Of Ph and Temperaturesupporting

confidence: 66%

Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3

Cheng¹,

He²,

Sun³

et al. 2015

Journal of Microbiology and Biotechnology

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“…Similar fibrinolytic enzymes are also secreted by Bacillus species and they have been purified. Their properties, including molecular weight, optimum pH and temperature, stability, and substrate specificity, have been reported [1,36]. Nattokinase is produced as a preproenzyme and processed into an active enzyme with 275 amino acids.…”

Section: Introductionmentioning

confidence: 99%

Characterization of AprE176, a Fibrinolytic Enzyme from Bacillus subtilis HK176

Jeong¹,

Heo²,

Park³

et al. 2015

Journal of Microbiology and Biotechnology

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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.

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“…Microbial proteases hydrolyze and inactivate host antibodies (Mistry & Stockley, ), complement proteins (Honda‐Ogawa et al ., ), and antimicrobial peptides (Thomassin et al ., ). Bacterial proteases also degrade host fibrin (Yeo et al ., ) and extracellular matrix proteins (Ruggiero et al ., ) which retard pathogen spread during infection.…”

Section: Introductionmentioning

confidence: 99%

EnterohemorrhagicEscherichia coliOmpT regulates outer membrane vesicle biogenesis

Premjani

Tilley

Gruenheid

et al. 2014

FEMS Microbiol Lett

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Enterohemorrhagic Escherichia coli (EHEC) infection from food or water often results in severe diarrheal disease and is a leading cause of death globally. Outer membrane vesicles (OMVs) secreted from E. coli induce lethality in mice. The omptin outer membrane protease OmpT from E. coli inactivates antimicrobial peptides and may enhance colonization of the uroepithelium, but its precise function remains unclear. Given OmpT is an outer membrane protease, we hypothesized it may have a role in OMV biogenesis. To further characterize the effect of OmpT on OMV production, a genetic approach using wild type, an ompT deletion mutant and an ompT overexpressing construct in EHEC were employed. ompT gene deletion markedly decreased OMV production and stainable lipid but increased vesicle diameter. Conversely, ompT overexpression profoundly increased OMV biogenesis but decreased stainable lipid, protein content, and vesicle diameter. Alterations in EHEC ompT gene expression have an impact on the biogenesis, composition, and size of OMVs. Changes in ompT gene expression may dynamically alter OMV formation, composition, and diameter in response to different host environments and contribute to cell-free intercellular communication to enhance bacterial growth and survival.

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Biochemical analysis of a fibrinolytic enzyme purified from Bacillus subtilis strain A1

Cited by 11 publications

References 21 publications

Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3

Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Streptomyces sp. P3

Characterization of AprE176, a Fibrinolytic Enzyme from Bacillus subtilis HK176

EnterohemorrhagicEscherichia coliOmpT regulates outer membrane vesicle biogenesis

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