Cloning, Heterologous Expression and Properties of a Recombinant Active Turnip Peroxidase
Authors:
- 18 May 2011
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of Agricultural and Food Chemistry
- Vol. 59 (13) , 7120-7126
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.Keywords
This publication has 31 references indexed in Scilit:
- Rachiplusia nu larva as a biofactory to achieve high level expression of horseradish peroxidaseBiotechnology Letters, 2011
- Peroxidase in Chemical and Biochemical AnalysisAnalytical Letters, 2010
- Effects of mutations in the helix G region of horseradish peroxidaseBiochimie, 2008
- Chemical Modification of Turnip Peroxidase with Methoxypolyethylene Glycol Enhances Activity and Stability for Phenol Removal Using the Immobilized EnzymeJournal of Agricultural and Food Chemistry, 2008
- High-level expression and purification of recombinant horseradish peroxidase isozyme C in SF-9 insect cell cultureProcess Biochemistry, 2005
- Isolation and Thermal Characterization of an Acidic Isoperoxidase from Turnip RootsJournal of Agricultural and Food Chemistry, 2003
- A peroxidase isoenzyme secreted by turnip (Brassica napus) hairy‐root cultures: inactivation by hydrogen peroxide and application in diagnostic kitsBiotechnology and Applied Biochemistry, 2002
- Estimation of the number of α‐helical and β‐strand segments in proteins using circular dichroism spectroscopyProtein Science, 1999
- Effect of single‐point mutations Phe41→ His and Phe143→ Glu on folding and catalytic properties of recombinant horseradish peroxidase expressed in E. coliFEBS Letters, 1994
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976