期刊
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
卷 25, 期 1, 页码 89-97出版社
KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY
DOI: 10.4014/jmb.1409.09087
关键词
Bacillus subtilis; fibrinolytic enzyme; error-prone PCR; thermostability
资金
- Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET), Ministry of Agriculture, Food and Rural Affairs, Republic of Korea [112066-03-SB010]
- MOE, Republic of Korea
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 degrees 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 (k(cat)/K-m) 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 degrees 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.
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