Purification, cloning, expression, and biochemical characterization of a monofunctional catalase, KatP, from Pigmentiphaga sp. DL-8.

Abstract Catalases are essential components of the cellular equipment used to cope with oxidative stress. The monofunctional catalase KatP was purified from Pigmentiphaga sp. using ammonium sulfate precipitation (ASP), diethylaminoethyl ion exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC). The purified catalase formed polymer with an estimated monomer molecular mass of 54 kDa, which were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and zymogram analysis. KatP exhibited a specific catalytic activity of 73,000 U/mg, which was higher than that of catalase-1 of Comamonas terrigena N3H (55,900 U/mg). Seven short tryptic fragments of this catalase were obtained by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS/MS), and the gene, katP , was cloned by PCR amplification and overexpressed in Escherichia coli BL21 (DE3). Based on the complete amino acid sequence, KatP was identified as a clade 3 monofunctional catalase. The specific activities of recombinant KatP for hydrogen peroxide (690,000 U/mg) increased 9-fold over that of the parent strain. The K m and V max of recombinant KatP were 9.48 mM and 81.2 mol/min mg, respectively. The optimal pH and temperature for KatP were 7.0 and 37 °C, respectively, and the enzyme displayed abroad pH-stable range of 4.0–11.0. The enzyme was inhibited by Zn 2+ , Cu 2+ , Cr 2+ , and Mn 2+ , whereas Fe 3+ and Mg 2+ stimulated KatP enzymatic activity. Interestingly, the catalase activity of recombinant KatP displayed high stability under different temperature and pH conditions, suggesting that KatP is a potential candidate for the production of catalase.