The dipeptidyl carboxypeptidase of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue: overproduction and molecular characterization of the recombinant enzyme |
| |
Authors: | Hsiang-Ling Chen Chen-Tien Chang Long-Liu Lin Tein-Yu Li Huei-Fen Lo |
| |
Institution: | (1) Department of Food and Nutrition, Providence University, Shalu, Taichung, 433, Taiwan, ROC;(2) Department of Food and Nutrition, Hungkuang University, Shalu, Taichung, 433, Taiwan, ROC;(3) Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi, 600, Taiwan, ROC |
| |
Abstract: | To express Escherichia coli novablue dipeptidyl carboxypeptidase (EcDCP), the gene was amplified by PCR and cloned into the expression plasmid pQE-31 to yield pQE-EcDCP. His6-tagged EcDCP (His6-EcDCP) was over-expressed in E. coli M15 (pQE-EcDCP) as a soluble and active form under 0.05 mM IPTG induction at 26°C for 12 h. The recombinant enzyme was purified to homogeneity
by Ni2+-NTA resin and had a molecular mass of approximately 75 kDa. The temperature and pH optima for His6-EcDCP were 37°C and 7.0, respectively. In the presence of 200 mM NaCl, His6-EcDCP was stimulated by 1.5 fold. The K
M and k
cat values of the enzyme for N-benzoyl-l-glycyl-l-histidyl-l-leucine were 1.83 mM and 168.3 s−1, respectively. His6-EcDCP activity was dramatically inhibited by 10 mM EDTA, 0.25 mM 1.10-phenanthroline, and 2.5 mM DEPC, but it was not affected
by Ser, Asp, Lys, and Trp protease inhibitors. Analysis of His6-EcDCP by circular dichroism revealed that the secondary structures of the enzyme in 30 mM universal buffer (pH 7.0) were 17%
α-helix, 35% β-sheet and 47% random coil. Mid point of thermal transition was calculated to be 55°C for the recombinant enzyme. |
| |
Keywords: | Escherichia coli Dipeptidyl carboxypeptidase Overexpression Circular dichroism |
本文献已被 SpringerLink 等数据库收录! |
|