title: 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.
authors.affiliation: Division of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University, Jinju 660-701, Republic of Korea.
authors.forename: Seon-Ju
authors.initials: SJ
authors.lastname: Jeong
authors.forename: Kyeong
authors.initials: K
authors.lastname: Heo
authors.forename: Ji Yeong
authors.initials: JY
authors.lastname: Park
authors.forename: Kang Wook
authors.initials: KW
authors.lastname: Lee
authors.forename: Jae-Yong
authors.initials: JY
authors.lastname: Park
authors.forename: Sang Hoon
authors.initials: SH
authors.lastname: Joo
authors.forename: Jeong Hwan
authors.initials: JH
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D005342:Fibrinolysis / Q000235:genetics*; D008892:Milk; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D045730:Soy Foods / Q000382:microbiology*; D013696:Temperature
chemical_list: D005337:Fibrin; D005340:Fibrinogen; D057057:Serine Proteases; D005341:Fibrinolysin