Cloning, heterologous expression and properties of a recombinant active turnip peroxidase |
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| Authors: | Rodriguez-Cabrera Norma A Regalado C Garcia-Almendarez Blanca E |
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| Institution: | Facultad de Qui?mica, Universidad Auto?noma de Quere?taro, Quere?taro, 76010 Qro. Me?xico. |
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| Abstract: | Turnip (Brassica napus) roots peroxidase isoforms have been used in diagnostic kits and can also efficiently polymerize phenolic compounds from wastewaters. Heterologous expression of a turnip acidic peroxidase (BnPA) was investigated to increase availability of this widely used enzyme. The mature BnPA was ligated into the pET28a(+) vector and used to transform Escherichia coli Rosetta 2. Recombinant BnPA peroxidase was overexpressed and accumulated in inclusion bodies from which it was purified to homogeneity by immobilized metal affinity chromatography under denaturing conditions. Peroxidase activity was observed after a refolding process under oxidative conditions. The yield of pure recombinant BnPA was 29 mg L(-1) of culture with a specific activity of 981 ± 20 ABTS units mg(-1) at optimal conditions (pH 6, 45 °C). Recombinant BnPA showed similar kinetic properties compared to native turnip peroxidase, and its secondary structure evaluated by circular dichroism comprised 20% α-helix, 32% β-sheet and 48% random structure. Recombinant BnPA showed high yield and good kinetic properties which are key steps for future structure-function studies and biotechnological applications. |
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