Purification, enzymatic properties of a recombinant d-hydantoinase and its dissociation by zinc ion |
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Authors: | Ya-Wei Shi Li-Xi Niu Xia Feng Jing-Ming Yuan |
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Institution: | (1) Institute of Biotechnology, Key Laboratory of Chemical Biology and Molecular Engineering of National Ministry of Education, Shanxi University, 92 Wucheng Road, 030006 Taiyuan, P. R. China |
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Abstract: | Summary A d-hydantoinase was expressed in the soluble form by a recombinant E. coli strain, pE-HDT/E. coli BL21 in LB medium. The enzymatic activity of cultured cells reached 5.2–6.5 IU/ml culture at a cell turbidity of 10 at 600 nm.
The expressed enzyme was efficiently purified by three steps, ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic
interaction chromatography and Sephacryl S-200 size-exclusion chromatography. With the above purification process, the enzyme
was purified to more than 95% purity as estimated by SDS-PAGE. The overall recovery of enzymatic activity was 54.4% and the
specific activity for substrate dl-hydantoin achieved 48 U/mg. The purified enzyme appeared as a dimer with a molecular mass of 103 kDa, as measured by size-exclusion
chromatography. The enzyme was stable from pH 6 to 12 with an optimum pH at 9.5 The optimum temperature of the enzyme was
45 °C and it activity was rapidly lost over 55 °C. Divalent metal ions, including Co2+, Mn2+ and Ni 2+ ions obviously enhanced the enzymatic activity, while Zn2+ ion had a slight inhibitory effect. In addition, the dissociation of purified enzyme into its subunits occurred in the presence
of 1 mM Zn2+ ion. The effect of different metal ions on the d-hydantoinase activation/attenuation was discussed. |
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Keywords: | d-hydantoinase" target="_blank">d-hydantoinase dissociation properties purification zinc ion |
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