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KMID : 0545120140240070969
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Journal of Microbiology and Biotechnology
2014 Volume.24 No. 7 p.969 ~ p.978
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Overexpression of aprE2, a Fibrinolytic Enzyme Gene from Bacillus subtilis CH3-5, in Escherichia coli and the Properties of AprE2
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Jeong Seon-Ju
Cho Kye-Man
Lee Chang-Kwon
Kim Gyoung-Min
Shin Jung-Hye
Kim Jong-Sang
Kim Jeong-Hwan
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Abstract
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The aprE2 gene with its prosequence from Bacillus subtilis CH3-5 was overexpressed in Escherichia coli BL21(DE3) by using plasmid pET26b(+). After IPTG induction, active and mature AprE2 was produced when cells were grown at 20°C, whereas inactive and insoluble enzyme was produced in a large amount when cells were grown at 37°C. The insoluble fraction was resuspended with 6 M guanidine-HCl and dialyzed against 2 M Tris-HCl (pH 7.0) or 0.5 M sodium acetate (pH 7.0) buffer. Then active AprE2 was regenerated and purified by a Ni-NTA column. Purified AprE2 from the soluble fraction had a specific activity of 1,069.4 ± 42.4 U/mg protein, higher than that from the renatured insoluble fraction. However, more active AprE2 was obtained by renaturation of the insoluble fraction. AprE2 was most stable at pH 7 and 40°C, respectively. The fibrinolytic activity of AprE2 was inhibited by PMSF, but not by EDTA and metal ions. AprE2 degraded Aα and Bβ chains of fibrinogen quickly, but not the γ-chain. AprE2 exhibited the highest specificity for N-succinyl-Ala-Ala-Pro-Phe-pNA. The Km and kcat/Km of AprE2 was 0.56 mM and 3.10 × 104 S-1 M-1, respectively.
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KEYWORD
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fibrinolytic enzyme, aprE2, Bacillus subtilis, overexpression
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