Journal of Microbiology and Biotechnology
The Korean Society for Microbiology and Biotechnology publishes the Journal of Microbiology and Biotechnology.

2015 ; Vol.25-1: 89~97

AuthorSeon-Ju Jeong, Kyeong Heo, Ji Yeong Park, Kang Wook Lee, Jae-Yong Park, Sang Hoon Joo, Jeong Hwan Kim
Place of dutyDivision of Applied Life Science (BK21 Plus), Graduate School, Gyeongsang National University, Jinju 660-701, Republic of Korea
TitleCharacterization of AprE176, a Fibrinolytic Enzyme from Bacillus subtilis HK176
PublicationInfo J. Microbiol. Biotechnol.2015 ; Vol.25-1
AbstractBacillus 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 176th residue of M179, threonine, was located near the cation-binding site compared with the wild type. This probably caused tight binding of M179 with Ca2+, which increased the thermostability of M179.
Key_wordBacillus subtilis, fibrinolytic enzyme, error-prone PCR, thermostability
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